AU779443B2 - Fen-1 endonucleases, mixtures and cleavage methods - Google Patents

Description

S&F Ref: 465707D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Third Wave Technologies, Inc.

502 South Rosa Road Madison Wisconsin 53719 United States of America Michael W. Kaiser, Victor I. Lyamichev, Natasha Lyamicheva Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Fen-1 Endonucleases, Mixtures and Cleavage Methods The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c FEN-I ENDONUCLEASES, MIXTURES AND CLEAVAGE METHODS FIELD OF THE INVENTION The present invention relates to means for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further provides novel methods and devices for the separation of nucleic acid molecules based by charge.

BACKGROUND OF THE INVENTION The detection and characterization of specific nucleic acid sequences and sequence variations has been utilized to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection, the presence of variants or alleles of mammalian genes associated with disease and cancers and the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations.

Various methods are known to the art which may be used to detect and characterize _20 specific nucleic acid sequences and sequence variants. Nonetheless, as nucleic acid sequence data of the human genome, as well as the genomes of pathogenic organisms accumulates, the demand for fast, reliable, cost-effective and user-friendly tests for the detection of specific nucleic acid sequences continues to grow. Importantly, these tests must be able to create a ,detectable signal from samples which contain very few copies of the sequence of interest.

The following discussion examines two levels of nucleic acid detection assays currently in use: I. Signal Amplification Technology for detection of rare sequences; and II. Direct °Detection Technology for detection of higher copy number sequences.

-1- I. Signal Amplification Technology Methods For Amplification The "Polymerase Chain Reaction" (PCR) comprises the first generation of methods for nucleic acid amplification. However, several other methods have been developed that employ the same basis of specificity, but create signal by different amplification mechanisms. These methods include the "Ligase Chain Reaction" (LCR), "Self-Sustained Synthetic Reaction" (3SR/NASBA), and "QP-Replicase" (Qp).

Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR), as described in U.S. Patent Nos. 4,683.195 and 4,683,202 to Mullis and Mullis et al. (the disclosures of which are hereby incorporated by reference), describe a method for increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification. This technology provides one approach to the problems of low target sequence concentration. PCR can be used to directly increase the concentration of the target to an easily detectable level. This process for amplifying the target sequence involves introducing a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, 20 hybridization, and polymerase extension can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.

The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable ~parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified." Ligase Chain Reaction (LCR or LAR) The ligase chain reaction (LCR; sometimes referred to as "Ligase Amplification Reaction" (LAR) described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR 30 Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed into a well-recognized alternative method for amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. Segev, PCT Public. No. W09001069 Al (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

Self-Sustained Synthetic Reaction (3SR/NASBA) The self-sustained sequence replication reaction (3SR) (Guatelli et al., Proc. Natl.

Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitro amplification system (Kwok el al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest. In a cocktail of 20 enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA 200-300 base pairs).

Q-Beta Replicase SIn this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for QP replicase. A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 0 This prevents the use of high temperature as a means of achieving specificity as in the LCR. the ligation event can be used to detect a mutation at the junction site, but not elsewhere.

Table 1 below, lists some of the features desirable for systems useful in sensitive nucleic acid diagnostics, and summarizes the abilities of each of the major amplification methods (See also, Landgren, Trends in Genetics 9:199 [1993]).

A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Qp systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature >55 0 Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons. PCR and LCR currently dominate the research field in detection technologies.

TABLE 1 FEATURE METHOD: PCR LCR PCR& 3SR Q# LCR NASBA Amplifies Target Recognition of Independent Sequences Required Performed at High Temp. Operates at Fixed Temp. Exponential Amplification Generic Signal Generation Easily Automatable The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as: y, where is the mean efficiency (percent copied in each cycle), is the number of cycles, and is the overall efficiency, or yield of the reaction (Mullis, PCR Methods Applic., 1:1 [1991]). If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100%. If 20 cycles of PCR are performed, then the yield will be 220, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85%, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material. In other words, a PCR running at 85% efficiency will yield only 21% as much final product, compared to a reaction running at 100% efficiency. A reaction that is reduced to 50% mean efficiency will yield less than 1% of the possible product.

In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield.

At 50% mean efficiency, it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products.

Also, many variables can influence the mean efficiency of PCR, including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA DNA spilled onto lab surfaces) or cross-contamination is also a major consideration. Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator. The o- °olaboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise S temperature cycling.

Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method for the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence. This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect (Kwok et al., Nucl. Acids Res., 18:999 [1990]).) A similar 3'-mismatch strategy is used with greater effect to prevent ligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.

I1. Direct Detection Technology When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the S* products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Traditional methods of direct detection including Northem and Southern blotting and RNase protection assays usually require the use of radioactivity and are not amenable to automation. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats.

Two examples are the "Cycling Probe Reaction" (CPR), and "Branched DNA" (bDNA) The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142 [1990]), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may be carried through sample preparation.

Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264 (1987), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry to 40 labels alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.

While both of these methods have the advantages of direct detection discussed above, neither the CPR or bDNA methods can make use of the specificity allowed by the requirement of independent recognition by two or more probe (oligonucleotide) sequences, as is common in the signal amplification methods described in section I. above. The requirement that two oligonucleotides must hybridize to a target nucleic acid in order for a detectable signal to be generated confers an extra measure of stringency on any detection assay. Requiring two oligonucleotides to bind to a target nucleic acid reduces the chance that false "positive" results will be produced due to the non-specific binding of a probe to the target. The further requirement that the two oligonucleotides must bind in a specific orientation relative to the target, as is required in PCR, where oligonucleotides must be oppositely but appropriately oriented such that the DNA polymerase can bridge the gap between the two oligonucleotides in both directions, further enhances specificity of the o. detection reaction. However, it is well known to those in the art that even though PCR utilizes two oligonucleotide probes (termed primers) "non-specific" amplification amplification of sequences not directed by the two primers used) is a common artifact. This 20 is in part because the DNA polymerase used in PCR can accommodate very large distances, measured in nucleotides, between the oligonucleotides and thus there is a large window in which non-specific binding of an oligonucleotide can lead to exponential amplification of inappropriate product. The LCR, in contrast, cannot proceed unless the oligonucleotides used are bound to the target adjacent to each other and so the full benefit of the dual oligonucleotide hybridization is realized.

An ideal direct detection method would combine the advantages of the direct detection assays easy quantification and minimal risk of carry-over contamination) with the specificity provided by a dual oligonucleotide hybridization assay.

NOV. 2004 12:25 SPRUSON FERGUSON NO. 9150 F. 8/11 8 Summary of the Invention According to a first embodiment of the invention, there is provided a mixture comprising; i) a first stiicurv-specific nuciease, wherein said first nuclease comprises a purified archaeal FEN-1 endonuclease, and wherein said FEN-1 endonuclease includes s the amino acid sequence VFDG; and ii) a purified archaeal 5' nuclease.

According to a second embodiment of the invention, there is provided a nucleic acid treatment kit comprising: a) a composition comprising purified archaeal FEN-1 endonuclease wherein said FEN-1 endonuclease includes the amino acid sequence VFDG; 0t b) a first oligonucleotide comprising a 5' portion complementary to a first portion of a target nucleic acid; and c) a second oligonucleotide comprising: a 5' portion complementary to a second portion of said target nucleic acid downstream of and contiguous to said first portion; and a 3' portion.

is According to a third embodiment of the invention, there is provided a kit comprising a chimerical FEN-1 endonuclease, wherein said chimerical endonuclease comprises at least a portion of an archaeal FEN-1 endonuclease.

The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. In one embodiment, the means for cleaving is a cleaving 20o enzyme comprising 5' nucleases derived from thermostable DNA polymerases. These polymerases form the basis of a novel method of detection of specific nucleic acid sequences. The present invention contemplates use of novel detection methods for various uses, including, but not limited to clinical diagnostic purposes.

In one embodiment, the present invention contemplates a DNA sequence encoding a DNA polymerase altered in sequence a "mutant" DNA polymerase) relative to the native sequence, such that it exhibits altered DNA synthetic activity from that of the native "wild type") DNA polymerase. It is preferred that the encoded DNA polymerase is altered such that it exhibits reduced synthetic activity compared to that of the native DNA polymerase. In this manner, the enzymes of the invention are S* 30 predominantly 5' nucleases and are capable of cleaving nucleic acids in a structure- S. specific manner in the absence of interfering synthetic activity.

(R:\LlBFFJ06779.doc:NSS COMS ID No: SBMI-01013091 Received by IP Australia: Time 12:32 Date 2004-11-25 Importantly, the 5' nucleases of the present invention are capable of cleaving linear duplex structures to create single discrete cleavage products. These linear structures areeither 1) not cleaved by the wild type enzymes (to any significant degree), or 2) are cleaved by the wild type enzymes so as to create multiple products. This characteristic of the 5' nucleases has been found to be a consistent property of enzymes derived in this manner from thermostable polymerases across eubacterial thermophilic species.

It is not intended that the invention be limited by the nature of the alteration necessary to render the polymerase synthesis-deficient. Nor is it intended that the invention be limited by the extent of the deficiency. The present invention contemplates various structures, including altered structures (primary, secondary, etc.), as well as native structures, that may be inhibited by synthesis inhibitors.

Where the polymerase structure is altered, it is not intended that the invention be limited by the means by which the structure is altered. In one embodiment, the alteration of the native DNA sequence comprises a change in a single nucleotide. In another 15 embodiment, the alteration of the native DNA sequence comprises a deletion of one or more nucleotides. In yet another embodiment, the alteration of the native DNA sequence comprises an insertion of one or more nucleotides. It is contemplated that the change in DNA sequence may manifest itself as change in amino acid sequence.

[R:\LIBFF]06751 .doc:NSS The present invention contemplates structure-specific nucleases from a variety of sources, including mesophilic, psychrophilic, thermophilic, and hyperthermophilic organisms.

The preferred structure-specific nucleases are thermostable. Thermostable structure-specific nucleases are contemplated as particularly useful in that they operate at temperatures where nucleic acid hybridization is extremely specific, allowing for allele-specific detection (including single-base mismatches). In one embodiment, the thermostable structure-specific are thermostable 5' nucleases which are selected from the group consisting of altered polymerases derived from the native polymerases of Thermus species, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limited to the use of thermostable 5' nucleases. Thermostable structurespecific nucleases from the FEN-1, RAD2 and XPG class of nucleases are also preferred.

The present invention provides a composition comprising a cleavage structure, the cleavage structure comprising: a) a target nucleic acid, the target nucleic acid having a first region, a second region, a third region and a fourth region, wherein the first region is located adjacent to and downstream from the second region, the second region is located adjacent to and downstream from the third region and the third region is located adjacent to and downstream from the fourth region; b) a first oligonucleotide complementary to the fourth region of the target nucleic acid; c) a second oligonucleotide having a 5' portion and a 3' portion wherein the 5' portion of the second oligonucleotide contains a sequence .20 complementary to the second region of the target nucleic acid and wherein the 3' portion of the second oligonucleotide contains a sequence complementary to the third region of the target nucleic acid; and d) a third oligonucleotide having a 5' portion and a 3' portion wherein the 5' portion of the third oligonucleotide contains a sequence complementary to the first region of the target nucleic acid and wherein the 3' portion of the third oligonucleotide contains a sequence complementary to the second region of the target nucleic acid.

The present invention is not limited by the length of the four regions of the target nucleic acid. In one embodiment, the first region of the target nucleic acid has a length of 11 to 50 nucleotides. In another embodiment, the second region of the target nucleic acid has a Slength of one to three nucleotides. In another embodiment, the third region of the target 30 nucleic acid has a length of six to nine nucleotides. In yet another embodiment, the fourth region of the target nucleic acid has a length of 6 to 50 nucleotides.

-9- The invention is not limited by the nature or composition of the of the first, second.

third and fourth oligonucleotides; these oligonucleotides may comprise DNA, RNA. PNA and combinations thereof as well as comprise modified nucleotides, universal bases, adducts. etc.

Further, one or more of the fi-st, Sccund, third and the fourth oiigonucleotides may contain a dideoxynucleotide at the 3' terminus.

In one preferred embodiment, the target nucleic acid is not completely complementary to at least one of the first, the second, the third and the fourth oligonucleotides. In a particularly preferred embodiment, the target nucleic acid is not completely complementary to the second oligonucleotide.

As noted above, the present invention contemplates the use of structure-specific nucleases in detection methods. In one embodiment, the present invention provides a method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising: a) providing: i) a cleavage means, ii) a source of target nucleic acid, the target nucleic acid having a first region, a second region, a third region and a fourth region, wherein the first region is located adjacent to and downstream from the second region, the second region is located adjacent to and downstream from the third region and the third region is located adjacent to and downstream from the fourth region; iii) a first oligonucleotide complementary to the fourth region of the target nucleic acid; iv) a second •oligonucleotide having a 5' portion and a 3' portion wherein the 5' portion of the second oligonucleotide contains a sequence complementary to the second region of the target nucleic acid and wherein the 3' portion of the second oligonucleotide contains a sequence complementary to the third region of the target nucleic acid; iv) a third oligonucleotide having a 5' and a 3' portion wherein the 5' portion of the third oligonucleotide contains a sequence complementary to the first region of the target nucleic acid and wherein the 3' portion of the third oligonucleotide contains a sequence complementary to the second region of the target nucleic acid; b) mixing the cleavage means, the target nucleic acid, the first oligonucleotide, the second oligonucleotide and the third oligonucleotide to create a reaction mixture under reaction conditions such that the first oligonucleotide is annealed to the fourth region of the target nucleic acid and wherein at least the 3' portion of the second oligonucleotide is 30 annealed to the target nucleic acid and wherein at least the 5' portion of the third oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure and wherein cleavage of the cleavage structure occurs to generate non-target cleavage products, 10 each non-target cleavage product having a 3'-hydroxyl group; and c) detecting the non-target cleavage products.

The invention is not limited by the nature of the target nucleic acid. In one embodiment, the target nucleic acid comprises single-stranded DNA. In another embodiment, the target nucleic acid comprises double-stranded DNA and prior to step the reaction mixture is treated such that the double-stranded DNA is rendered substantially single-stranded.

In another embodiment, the target nucleic acid comprises RNA and the first and second oligonucleotides comprise DNA.

The invention is not limited by the nature of the cleavage means. In one embodiment, the cleavage means is a structure-specific nuclease; particularly preferred structure-specific nucleases are thermostable structure-specific nucleases. In one preferred embodiment, the thermostable structure-specific nuclease is encoded by a DNA sequence selected from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30, 31, 101, 106, 110, 114, 129. 131, 132, 137, 140, 141, 142, 143, 144, 145, 147, 150, 151, 153, 155, 156, 157, 158, 161, 163, 178, 180, and 182.

In another preferred embodiment, the thermostable structure-specific nuclease is a nuclease from the FEN-1/RAD2/XPG class of nucleases. In another preferred embodiment the thermostable structure specific nuclease is a chimeric nuclease.

In an alternative preferred embodiment, the detection of the non-target cleavage 20 products comprises electrophoretic separation of the products of the reaction followed by visualization of the separated non-target cleavage products.

In another preferred embodiment, one or more of the first, second, and third oligonucleotides contain a dideoxynucleotide at the 3' terminus. When dideoxynucleotidecontaining oligonucleotides are employed, the detection of the non-target cleavage products preferably comprises: a) incubating the non-target cleavage products with a templateindependent polymerase and at least one labeled nucleoside triphosphate under conditions such that at least one labeled nucleotide is added to the 3'-hydroxyl group of the non-target cleavage products to generate labeled non-target cleavage products; and b) detecting the S* presence of the labeled non-target cleavage products. The invention is not limited by the nature of the template-independent polymerase employed; in one embodiment, the templateindependent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT or polyA polymerase are employed in the detection step, the second oligonucleotide may contain a 5' end label, the 5' end label 11 being a different label than the label present upon the labeled nucleoside triphosphate. The invention is not limited by the nature of the 5' end label; a wide variety of suitable 5' end labels are known to the art and include biotin, fluorescein, tetrachlorofluorescein, lhxachiorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage products comprises: a) incubating the non-target cleavage products with a template-independent polymerase and at least one nucleoside triphosphate under conditions such that at least one nucleotide is added to the 3'-hydroxyl group of the non-target cleavage products to generate tailed non-target cleavage products; and b) detecting the presence of the tailed non-target cleavage products.

The invention is not limited by the nature of the template-independent polymerase employed: in one embodiment, the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT or polyA polymerase are employed in the detection step, the second oligonucleotide may contain a 5' end label. The invention is not limited by the nature of the 5' end label; a wide variety of suitable 5' end labels are known to the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In a preferred embodiment, the reaction conditions comprise providing a source of divalent cations; particularly preferred divalent cations are Mn* and Mg 2 ions.

The present invention further provides a method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising: a) providing: i) a cleavage means, ii) a source of target nucleic acid, the target nucleic acid having a first region, a second region and a third region, wherein the first region is located adjacent to and downstream from the second region and wherein the second region is located adjacent to and **downstream from the third region; iii) a first oligonucleotide having a 5' and a 3' portion wherein the 5' portion of the first oligonucleotide contains a sequence complementary to the second region of the target nucleic acid and wherein the 3' portion of the first oligonucleotide contains a sequence complementary to the third region of the target nucleic acid; iv) a second oligonucleotide having a length between eleven to fifteen nucleotides and further having a and a 3' portion wherein the 5' portion of the second oligonucleotide contains a sequence 30 complementary to the first region of the target nucleic acid and wherein the 3' portion of the second oligonucleotide contains a sequence complementary to the second region of the target nucleic acid; b) mixing the cleavage means, the target nucleic acid, the first oligonucleotide and the second oligonucleotide to create a reaction mixture under reaction conditions such that 12 at least the 3' portion of the first oligonucleotide is annealed to the target nucleic acid and wherein at least the 5' portion of the second oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure and wherein cleavage of the cleavage structure occurs to generate non-target cleavage products, each non-target cleavage product having a 3'hydroxyl group; and c) detecting the non-target cleavage products. In a preferred embodiment the cleavage means is a structure-specific nuclease, preferably a thermostable structure-specific nuclease.

The invention is not limited by the length of the various regions of the target nucleic acid. In a preferred embodiment, the second region of the target nucleic acid has a length between one to five nucleotidcs. In another preferred embodiment, one or more of the first and the second oligonucleotides contain a dideoxynucleotide at the 3' terminus. When dideoxynucleotide-containing oligonucleotides are employed, the detection of the non-target cleavage products preferably comprises: a) incubating the non-target cleavage products with a template-independent polymerase and at least one labeled nucleoside triphosphate under conditions such that at least one labeled nucleotide is added to the 3'-hydroxyl group of the non-target cleavage products to generate labeled non-target cleavage products; and b) detecting the presence of the labeled non-target cleavage products. The invention is not limited by the nature of the template-independent polymerase employed; in one embodiment, the template-independent polymerase is selected from the group consisting of terminal 20 deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT or polyA polymerase is employed in the detection step, the second oligonucleotide may contain a 5' end label, the end label being a different label than the label present upon the labeled nucleoside triphosphate. The invention is not limited by the nature of the 5' end label; a wide variety of suitable 5' end labels are known to the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

In another embodiment, detecting the non-target cleavage products comprises: a) "incubating the non-target cleavage products with a template-independent polymerase and at least one nucleoside triphosphate under conditions such that at least one nucleotide is added to the 3'-hydroxyl group of the non-target cleavage products to generate tailed non-target 30 cleavage products; and b) detecting the presence of the tailed non-target cleavage products.

The invention is not limited by the nature of the template-independent polymerase employed; in one embodiment, the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. When TdT 13 or polyA polymerase are employed in the detection step, the second oligonucleotide may contain a 5' end label. The invention is not limited by the nature of the 5' end label; a wide variety of suitable 5' end labels are known to the art and include biotin, fluorescein, tetrachlnrnflllnr rr, n h v -ln rI terachlro...ores heach ucre in, Cy3 amidite, Cy5 amidJ i and digoxigenin.

The novel detection methods of the invention may be employed for the detection of target DNAs and RNAs including, but not limited to, target DNAs and RNAs comprising wild type and mutant alleles of genes, including genes from humans or other animals that are or may be associated with disease or cancer. In addition, the methods of the invention may be used for the detection of and/or identification of strains of microorganisms, including bacteria, fungi, protozoa, ciliates and viruses (and in particular for the detection and identification of RNA viruses, such as HCV).

The present invention further provides improved enzymatic cleavage means. In one embodiment, the present invention provides a thermostable structure-specific nuclease having an amino acid sequence selected from the group consisting of SEQ ID NOS:102, 107, 130, 132, 179, 181, 183, 184, 185, 186, 187, and 188. In another embodiment, the nuclease is encoded by a DNA sequence selected from the group consisting of SEQ ID NOS:101, 106, 129 131, 178, 180, and 182.

The present invention also provides a recombinant DNA vector comprising DNA having a nucleotide sequence encoding a structure-specific nuclease, the nucleotide sequence 20 selected from the group consisting of SEQ ID NOS:101, 106, 129 131, 137, 140, 141, 142, 143, 144, 145, 147, 150, 151, 153, 155, 156, 157, 158, 161, 163, 178, 180, and 182. In a preferred embodiment, the invention provides a host cell transformed with a recombinant DNA vector comprising DNA having a nucleotide sequence encoding a structure-specific S* nuclease, the nucleotide sequence selected from the group consisting of SEQ ID NOS:101, 25 106, 129, 131, 178, 180, and 182. The invention is not limited by the nature of the host cell employed. The art is well aware of expression vectors suitable for the expression of nucleotide sequences encoding structure-specific nucleases which can be expressed in a variety of prokaryotic and eukaryotic host cells. In a preferred embodiment, the host cell is an Escherichia coli cell.

30 The present invention provides purified FEN-1 endonucleases. In one embodiment, the present invention provides Pyrococcus woesei FEN-1 endonuclease. In a preferred embodiment, the purified Pyrococcus woesei FEN-I endonuclease has a molecular weight of 14about 38.7 kilodaltons (the molecular weight may be conveniently estimated using SDS- PAGE as described in Ex. 28).

The present invention further provides an isolated oligonucleotide encoding a Pyrococcus woesei FEN-1 endonuclease, the oligonucleotide having a region capable of hybridizing to an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:116-119. In a preferred embodiment, the oligonucleotide encoding the purified Pyrococcus woesei FEN-1 endonuclease is operably linked to a heterologous promoter. The present invention is not limited by the nature of the heterologous promoter employed; in a preferred embodiment, the heterologous promoter is an inducible promoter (the promoter chosen will depend upon the host cell chosen for expression as is known in the art). The invention is not limited by the nature of the inducible promoter employed. Preferred inducible promoter include the X-PL promoter, the lac promoter, the trp promoter and the trc promoter.

In another preferred embodiment, the invention provides a recombinant DNA vector comprising an isolated oligonucleotide encoding a Pyrococcus woesei (Pwo) FEN-I endonuclease, the oligonucleotide having a region capable of hybridizing to an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:116-119. Host cells transformed with these recombinant vectors are also provided. In a preferred embodiment, the invention provides a host cell transformed with a recombinant DNA vector comprising DNA having a o* 20 region capable of hybridizing to an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:116-119; these vectors may further comprise a heterologous promoter operably linked to the Pwo FEN-l-encoding polynucleotides. The invention is not limited by the nature of the host cell employed. The art is well aware of expression vectors suitable for the expression of Pwo FEN-l-encoding polynucleotides which can be expressed in a variety of prokaryotic and eukaryotic host cells. In a preferred embodiment, the host cell is an Escherichia coli cell.

In yet another embodiment, the invention provides an isolated oligonucleotide comprising a gene encoding a Pyrococcus woesei FEN-1 endonuclease having a molecular weight of about 38.7 kilodaltons. In another embodiment, the encoding a Pyrococcus woesei 30 FEN-1 endonuclease is operably linked to a heterologous promoter. The present invention is not limited by the nature of the heterologous promoter employed; in a preferred embodiment, the heterologous promoter is an inducible promoter (the promoter chosen will depend upon the host cell chosen for expression as is known in the art). The invention is not limited by the nature of the inducible promoter employed. Preferred inducible promoter include the k-PL promoter, the tac promoter, the trp promoter and the trc promoter.

The invention further provides recombinant DNA vectors comprising DNA having a nucleotide sequence encoding FEN-i endonucieases. In one preferred embodiment, the present invention provides a Pyrococcus woesei FEN-I endonuclease having a molecular weight of about 38.7 kilodaltons. Still further, a host cell transformed with a recombinant DNA vector comprising DNA having a nucleotide sequence encoding FEN-I endonuclease.

In a preferred embodiment, the host cell is transformed with a recombinant DNA vector comprising DNA having a nucleotide sequence encoding a Pyrococcus woesei FEN-1 endonuclease having a molecular weight of about 38.7 kilodaltons is provided. The invention is not limited by the nature of the host cell employed. The art is well aware of expression vectors suitable for the expression of Pwo FEN-1-encoding polynucleotides which can be expressed in a variety of prokaryotic and eukaryotic host cells. In a preferred embodiment, the host cell is an Escherichia coli cell.

Thus, the present invention provides multiple purified FEN-1 endonucleases, both purified native forms of the endonucleases, as well as recombinant endonucleases. In preferred embodiments, the purified FEN-I endonucleases are obtained from archaebacterial or eubacterial organisms. In particularly preferred embodiments, the FEN-I endonucleases are obtained from organisms selected from the group consisting of Archaeoglobus fulgidus and I 20 Methanobacterium thermoautotrophicum. In a preferred embodiment, the purified FEN-I endonucleases have molecular weights of about 39 kilodaltons (the molecular weight may be conveniently estimated using SDS-PAGE as described in Ex. 28).

.The present invention further provides isolated oligonucleotides encoding Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum FEN-1 endonucleases, 25 the oligonucleotides each having a region capable of hybridizing to at least a portion of an oligonucleotide sequence, wherein the oligonucleotide sequence is selected from the group consisting of SEQ ID NOS:170, 171, 172, and 173. In some preferred embodiment, the oligonucleotides encoding the Archaeoglobusfulgidus and Methanobacterium thermoautoirophicum FEN-I endonucleases are operably linked to heterologous promoters.

.30 However, it is not intended that the present invention be limited by the nature of the heterologous promoter employed. It is contemplated that the promoter chosen will depend upon the host cell chosen for expression as is known in the art. In some preferred embodiments, the heterologous promoter is an inducible promoter. The invention is not 16 limited by the nature of the inducible promoter employed. Preferred inducible promoters include the X-PL promoter, the tac promoter, the trp promoter and the trc promoter.

In another preferred embodiment, the invention provides recombinant DNA vectors comprising isolated oligonucleotides encoding Archaeoglobus fulgidus or Methanobacterium thermoautolrophicum FEN-1 endonucleases, each oligonucleotides having a region capable of hybridizing to at least a portion of an oligonucleotide sequence, wherein the oligonucleotide sequence is selected from the group consisting of SEQ ID NOS:170, 171, 172, and 173. The present invention further provides host cells transformed with these recombinant vectors. In a preferred embodiment, the invention provides a host cell transformed with a recombinant DNA vector comprising DNA having a region capable of hybridizing to at least a portion of an oligonucleotide sequence, wherein the oligonucleotide sequence is selected from the group consisting of SEQ ID NOS:170, 171, 172 and 173. In some embodiments, these vectors may further comprise a heterologous promoter operably linked to the FEN-1-encoding polynucleotides. The invention is not limited by the nature of the host cell employed. The art is well aware of expression vectors suitable for the expression of FEN-1-encoding polynucleotides which can be expressed in a variety of prokaryotic and eukaryotic host cells.

In a preferred embodiment, the host cell is an Escherichia coli cell.

The present invention further provides chimeric structure-specific nucleases. In one *.oo Sembodiment, the present invention provides chimeric endonucleases comprising amino acid portions derived from the endonucleases selected from the group of FEN-1, XPG and RAD homologs. In a preferred embodiment, the chimeric endonucleases comprise amino acid portions derived from the FEN-I endonucleases selected from the group of Pyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei, Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. In a more preferred embodiment, the chimeric 25 FEN-1 endonucleases have molecular weights of about 39 kilodaltons (the molecular weight may be conveniently estimated using SDS-PAGE as described in Ex. 28).

The present invention further provides isolated oligonucleotides encoding chimeric endonucleases. In one embodiment, the oligonucleotides encoding the chimeric endonucleases comprise nucleic acid sequences derived from the genes selected from the group of FEN-1, 30 XPG and RAD homologs. In a preferred embodiment the oligonucleotides encoding the chimeric endonucleases comprise nucleic acid sequences derived from the genes encoding the FEN-1 endonucleases selected from the group of Pyrococcus furiosus, Methanococcus jannaschi. Pyrococcus woesei, Archaeoglobus fulgidus and Methanobacterium 17 .i ihermoautotrophicum. In a particularly preferred embodiment, the genes for the chimeric endonucleases are operably linked to heterologous promoters. The present invention is not limited by the nature of the heterologous promoter employed. It is contemplated that the promoter chosen will depend upon the host cel selected for cxpression, as is known in the art.

In preferred embodiments, the heterologous promoter is an inducible promoter. The invention is not limited by the nature of the inducible promoter employed. Preferred inducible promoter include the X-PL promoter, the tac promoter, the trp promoter and the tre promoter.

In another preferred embodiment, the invention provides recombinant DNA vectors comprising isolated oligonucleotides encoding the chimeric endonucleases described above. In one embodiment, the recombinant DNA vectors comprise isolated oligonucleotides encoding nucleic acid sequences derived from the genes selected from the group of FEN-1, XPG and RAD homologs. In a preferred embodiment, the recombinant DNA vectors comprise isolated oligonucleotides encoding the chimeric endonucleases comprising nucleic acid sequences derived from the genes encoding the FEN-1 endonucleases selected from the group of Pyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei, Archaeoglobus fulgidus and Methanobacterium thermoaufotrophicum. These vectors may further comprise a heterologous promoter operably linked to the chimeric nuclease-encoding polynucleotides.

Host cells transformed with these recombinant vectors are also provided. The invention is not limited by the nature of the host cell employed. The art is well aware of S 20 expression vectors suitable for the expression of FEN-l-encoding polynucleotides which can be expressed in a variety of prokaryotic and eukaryotic host cells. In a preferred embodiment, the host cell is an Escherichia coli cell.

The present invention further provides mixtures comprising a first structure-specific nuclease, wherein the first nuclease consists of a purified FEN-1 endonuclease and a second 25 structure-specific nuclease. In preferred embodiments, the second structure-specific nuclease of the mixture is selected from the group consisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1, Methanococcus jannaschii FEN-1 endonuclease, "Methanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobus fulgidus FEN-1, and chimerical FEN-1 endonucleases. In alternative embodiments, the purified FEN-1 endonuclease of the mixture is selected from the group consisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease, Methanobacterium thermoautotrophicum FEN-I endonuclease, Archaeoglobus fulgidus FEN-1, and chimerical FEN-I endonucleases. In yet other preferred embodiments of 18 the mixture. the second nuclease is a 5' nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same nuclease activity of the wild-type DNA polymerase. In some preferred embodiments of the mixture, the second nuclease is selected from the group consisting of the Cleavase® BN enzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, Escherichia coli Exo III, Saccharomyces cerevisiae Radl/RadlO complex.

The present invention also provides methods for treating nucleic acid, comprising: a) providing a purified FEN-I endonuclease; and a nucleic acid substrate; b) treating the nucleic acid substrate under conditions such that the substrate forms one or more cleavage structures; and c) reacting the endonuclease with the cleavage structures so that one or more cleavage products are produced. In some embodiments, the purified FEN-I endonuclease is selected from the group consisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease, Melhanohacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobus fulgidus FEN-1, and chimerical FEN-1 endonucleases. In other embodiments, the method further comprises providing a structure-specific nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same 5' nuclease activity of the wild- 20 type DNA polymerase.

In alternative embodiments of the methods, a portion of the amino acid sequence of the second nuclease is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a eubacterial thermophile of the genus Thermus. In yet other embodiments, the thermophile is selected from the group consisting of Thermus aquaticus, 25 Thermusflavus and Thermus thermophilus. In some alternative embodiments, the structurespecific nuclease is selected from the group consisting of the Cleavase® BN enzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, Escherichia coli Exo III, Saccharomyces cerevisiae Radl/RadlO complex. In some preferred embodiments, the structure-specific nuclease is the Cleavase® BN nuclease. In yet other embodiments, the 30 nucleic acid of step is substantially single-stranded. In further embodiments, the nucleic acid is selected from the group consisting of RNA and DNA. In yet further embodiments, the nucleic acid of step is double stranded.

19- In other embodiments of the methods, the treating of step comprises: rendering the double-stranded nucleic acid substantially single-stranded; and exposing the single-stranded nucleic acid to conditions such that the single-stranded nucleic acid has secondary structure.

in some preferred embodiments, the double stranded nucleic acid is rendered substantially single-stranded by the use of increased temperature. In alternative preferred embodiments, the method further comprises the step of detecting the one or more cleavage products.

The present invention also provides methods for treating nucleic acid, comprising: a) providing: a first structure-specific nuclease consisting of a purified FEN-1 endonuclease in a solution containing manganese; and a nucleic acid substrate; b) treating the nucleic acid substrate with increased temperature such that the substrate is substantially single-stranded; c) reducing the temperature under conditions such that the single-stranded substrate forms one or more cleavage structures; d) reacting the cleavage means with the cleavage structures so that one or more cleavage products are produced; and e) detecting the one or more cleavage products. In some embodiments of the methods, the purified FEN-I endonuclease is selected from the group consisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-I endonuclease, Methanobacterium thermoautotrophicum FEN-I endonuclease, Archaeoglobus fulgidus FEN-I, and chimerical FEN-I endonucleases. In alternative embodiments, the methods further comprise providing a second structure-specific nuclease. In some preferred embodiments, the second nuclease is 20 selected from the group consisting of the Cleavase® BN enzyme, Thermus aquaticus DNA polymerase. Thermus thcrmophilus DNA polymerase, Escherichia coli Exo III, and the Saccharonmces cerevisiae Radl/RadlO complex. In yet other preferred embodiments, the .*second nuclease is a 5' nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same 5' nuclease activity of the wildtype DNA polymerase. In yet other embodiments, the nucleic acid is selected from the group consisting of RNA and DNA. In further embodiments, the nucleic acid of step is double stranded.

The present invention also provides nucleic acid treatment kits, comprising: a) a composition comprising at least one purified FEN-I endonuclease; and b) a solution containing manganese. In some embodiments of the kits, the purified FEN-1 endonuclease is selected from the group consisting Pyrococcus woesei FEN-I endonuclease, Pyrococcus furiosus FEN-I endonuclease, Methanococcus jannaschii FEN-I endonuclease, Methanobacterium thermoautotrophicum FEN- endonuclease, Archaeoglobus fulgidus FEN-1, and chimerical FEN-1 endonucleases. In other embodiments, the kits further comprise at least one second structure-specific nuclease. In some preferred embodiments, the second nuclease is a 5' nuclease derived from a thermostable DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same 5' nuclease activity of the wild-type DNA polymerase. In yet other embodiments of the kits, the portion of the amino acid-sequence of the second nuclease is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a eubacterial thermophile of the genus Thermus. In further embodiments, the thermophile is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus. In yet other preferred embodiments, the kits further comprise reagents for detecting the cleavage products.

DESCRIPTION OF THE DRAWINGS Figure IA provides a schematic of one embodiment of the detection method of the present invention.

Figure 1B provides a schematic of a second embodiment of the detection method of the present invention.

Figure 2 is a comparison of the nucleotide structure of the DNAP genes isolated from Thermus aquaticus (SEQ ID NO:1), Thermusflavus (SEQ ID NO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence (SEQ ID NO:7) is shown at the top of each row.

Figure 3 is a comparison of the amino acid sequence of the DNAP isolated from Thermus aquaticus (SEQ ID NO:4), Thermusflavus (SEQ ID NO:5), and Thermus thermophilus (SEQ ID NO:6); the consensus sequence (SEQ ID NO:8) is shown at the top of each row.

Figures 4A-G are a set of diagrams of wild-type and synthesis-deficient DNAPTaq genes.

Figure 5A depicts the wild-type Thermusflavus polymerase gene.

Figure 5B depicts a synthesis-deficient Thermusflavus polymerase gene.

S*Figure 6 depicts a structure which cannot be amplified using DNAPTaq.

-21 Figure 7 is a ethidium bromide-stained gel demonstrating attempts to amplify a bifurcated duplex using either DNAPTaq or DNAPStf the Stoffel fragment of DNAPTaq).

Fig ue 8 ;s ar autoradiogran- of a gel analyzing the cleavage of a bifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

Figures 9A-B are a set of autoradiograms of gels analyzing cleavage or lack of cleavage upon addition of different reaction components and change of incubation temperature during attempts to cleave a bifurcated duplex with DNAPTaq.

Figures 1OA-B are an autoradiogram displaying timed cleavage reactions, with and without primer.

Figures 11A-B are a set of autoradiograms of gels demonstrating attempts to cleave a bifurcated duplex (with and without primer) with various DNAPs.

Figures 12A shows the substrates and oligonucleotides used to test the specific cleavage of substrate DNAs targeted by pilot oligonucleotides.

Figure 12B shows an autoradiogram of a gel showing the results of cleavage reactions using the substrates and oligonucleotides shown Fig. 12A.

Figure 13A shows the substrate and oligonucleotide used to test the specific cleavage of a substrate RNA targeted by a pilot oligonucleotide.

Figure 13B shows an autoradiogram of a gel showing the results of a cleavage reaction using the substrate and oligonucleotide shown in Fig. 13A.

Figure 14 is a diagram of vector pTTQ18.

Figure 15 is a diagram of vector pET-3c.

Figure 16A-E depicts a set of molecules which are suitable substrates for cleavage by the 5' nuclease activity of DNAPs.

25 Figure 17 is an autoradiogram of a gel showing the results of a cleavage reaction run with synthesis-deficient DNAPs.

Figure 18 is an autoradiogram of a PEI chromatogram resolving the products of an assay for synthetic activity in synthesis-deficient DNAPTaq clones.

"Figure 19A depicts the substrate molecule used to test the ability of synthesis-deficient DNAPs to cleave short hairpin structures.

Figure 19B shows an autoradiogram of a gel resolving the products of a cleavage S* reaction run using the substrate shown in Fig. 19A.

Figure 20A shows the A- and T-hairpin molecules used in the trigger/detection assay.

-22- Figure 20B shows the sequence of the alpha primer used in the trigger/detection assay.

Figure 20C shows the structure of the cleaved A- and T-hairpin molecules.

Figure 20D depicts the complementarity between the A- and T-hairpin molecules.

Figure 21 provides the complete 206-mer duplex sequence employed as a substrate for the 5' nucleases of the present invention Figures 22A and B show the cleavage of linear nucleic acid substrates (based on the 206-mer of Figure 21) by wild type DNAPs and 5' nucleases isolated from Thermus aquaticus and Thermus flavus.

Figure 23 provides a detailed schematic corresponding to one embodiment of the detection method of the present invention.

Figure 24 shows the propagation of cleavage of the linear duplex nucleic acid structures of Figure 23 by the 5' nucleases of the present invention.

Figure 25A shows the "nibbling" phenomenon detected with the DNAPs of the present invention.

Figure 25B shows that the "nibbling" of Figure 25A is 5' nucleolytic cleavage and not phosphatase cleavage.

Figure 26 demonstrates that the "nibbling" phenomenon is duplex dependent.

Figure 27 is a schematic showing how "nibbling" can be employed in a detection assay.

20 Figure 28 demonstrates that "nibbling" can be target directed.

:Figure 29 provides a schematic drawing of a target nucleic acid with an invader :oligonucleotide and a probe oligonucleotide annealed to the target.

Figure 30 provides a schematic showing the S-60 hairpin oligonucleotide (SEQ ID with the annealed P-15 oligonucleotide (SEQ ID NO:41).

25 Figure 31 is an autoradiogram of a gel showing the results of a cleavage reaction run using the S-60 hairpin in the presence or absence of the P-15 oligonucleotide.

Figure 32 provides a schematic showing three different arrangements of target-specific •oligonucleotides and their hybridization to a target nucleic acid which also has a probe oligonucleotide annealed thereto.

Figure 33 is the image generated by a fluorescence imager showing that the presence of an invader oligonucleotide causes a shift in the site of cleavage in a probe/target duplex.

23 Figure 34 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run using the three target-specific oligonucleotides diagrammed in Figure 32.

Figure 3S i the imigp a f....oesci Figure is the image generated by a fluorescenc imager showing the products of invader-directed cleavage assays run in the presence or absence of non-target nucleic acid molecules.

Figure 36 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of decreasing amounts of target nucleic acid.

Figure 37 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence or absence of saliva extract using various thermostable 5' nucleases or DNA polymerases.

Figure 38 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run using various 5' nucleases.

Figure 39 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run using two target nucleic acids which differ by a single basepair at two different reaction temperatures.

Figure 40A provides a schematic showing the effect of elevated temperature upon the annealing and cleavage of a probe oligonucleotide along a target nucleic acid wherein the probe contains a region of noncomplementarity with the target.

Figure 40B provides a schematic showing the effect of adding an upstream oligonucleotide upon the annealing and cleavage of a probe oligonucleotide along a target nucleic acid wherein the probe contains a region of noncomplementarity with the target.

Figure 41 provides a schematic showing an arrangement of a target-specific invader 25 oligonucleotide (SEQ ID NO:50) and a target-specific probe oligonucleotide (SEQ ID NO:49) bearing a 5' Cy3 label along a target nucleic acid (SEQ ID NO:42).

Figure 42 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of KC1..-- •Figure 43 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of NaCI.

Figure 44 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of LiC1.

24 Figure 45 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of KGlu.

Figure 46 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of MnCI, or MgCI 2 Figure 47 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of CTAB.

Figure 48 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of PEG.

Figure 49 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of glycerol, Tween-20 and/or Nonidet- Figure 50 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing concentrations of gelatin in reactions containing or lacking KCI or LiCl.

Figure 51 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run in the presence of increasing amounts of genomic DNA or tRNA.

Figure 52 is the image generated by a fluorescence imager showing the products of 20 invader-directed cleavage assays run use a HCV RNA target.

:Figure 53 is the image generated by a fluorescence imager showing the products of invader-directed cleavage assays run using a HCV RNA target and demonstrate the stability of RNA targets under invader-directed cleavage assay conditions.

Figure 54 is the image generated by a fluorescence imager showing the sensitivity of oo° 25 detection and the stability of RNA in invader-directed cleavage assays run using a HCV RNA target.

Figure 55 is the image generated by a fluorescence imager showing thermal "degradation of oligonucleotides containing or lacking a 3' phosphate group.

Figure 56 depicts the structure of amino-modified oigonuceotids 70 and 74.

Figure 56 depicts the structure of amino-modified oligonucleotides 70 and 74.

Figure 57 depicts the structure of amino-modified oligonucleotide Figure 58 depicts the structure of amino-modified oligonucleotide 76.

Figure 59 is the image generated by a fluorescence imager scan of an IEF gel showing the migration of substrates 70, 70dp, 74, 74dp, 75, 75dp, 76 and 76dp.

25 Figure 60A provides a schematic showing an arrangement of a target-specific invader oligonucleotide (SEQ ID NO:61) and a target-specific probe oligonucleotide (SEQ ID NO:62) bearing a 5' Cy3 label along a target nucleic acid (SEQ ID NO:63).

Figure 60B is the image generated by a fluorescence imager showing the detection of specific cleavage products generated in an invasive cleavage assay using charge reversal charge based separation of cleavage products).

Figure 61 is the image generated by a fluorescence imager which depicts the sensitivity of detection of specific cleavage products generated in an invasive cleavage assay using charge reversal.

Figure 62 depicts a first embodiment of a device for the charge-based separation of oligonucleotides.

Figure 63 depicts a second embodiment of a device for the charge-based separation of oligonucleotides.

Figure 64 shows an autoradiogram of a gel showing the results of cleavage reactions run in the presence or absence of a primer oligonucleotide; a sequencing ladder is shown as a size marker.

Figures 65a-d depict four pairs of oligonucleotides; in each pair shown, the upper arrangement of a probe annealed to a target nucleic acid lacks an upstream oligonucleotide and the lower arrangement contains an upstream oligonucleotide.

Figure 66 shows the chemical structure of several positively charged heterodimeric ~DNA-binding dyes.

Figure 67 is a schematic showing alternative methods for the tailing and detection of specific cleavage products in the context of the InvaderTM-directed cleavage assay.

Figure 68 provides a schematic drawing of a target nucleic acid with an InvaderTM 25 oligonucleotide, a miniprobe, and a stacker oligonucleotide annealed to the target.

Figure 69 provides a space-filling model of the 3-dimensional structure of the T5 exonuclease.

*oo Figure 70 provides an alignment of the amino acid sequences of several FEN-1 nucleases including the Methanococcus jannaschii FEN-1 protein (MJAFEN1.PRO), the Pyrococcus furiosus FEN-1 protein (PFUFEN1.PRO), the human FEN-1 protein S (HUMFEN .PRO), the mouse FEN-I protein (MUSFEN1.PRO), the Saccharomyces cerevisiae YKL510 protein (YST510.PRO), the Saccharomyces cerevisiae RAD2 protein (YSTRAD2.PRO), the Shizosaccharomyces pombe RADI3 protein (SPORAD13.PRO), the 26human XPG protein (HUMXPG.PRO), the mouse XPG protein (MUSXPG.PRO), the Xenopus laevis XPG protein (XENXPG.PRO) and the C. elegans RAD2 protein (CELRAD2.PRO); portions of the amino acid sequence of some of these proteins were not shown in order to maximize the alignment between proteins. The numbers to the left of each line of sequence refers to the amino acid residue number; dashes represent gaps introduced to maximize alignment.

Figure 71 is a schematic showing the S-33 (SEQ ID NO:120) and 11-8-0 (SEQ ID NO:121) oligonucleotides in a folded configuration; the cleavage site is indicated by the arrowhead.

Figure 72 shows a Coomassie stained SDS-PAGE gel showing the thrombin digestion of Cleavase® BN/thrombin.

Figure 73 is the image generated by a fluorescence imager showing the products produced by the cleavage of the S-60 hairpin using Cleavase® BN/thrombin (before and after thrombin digestion).

Figure 74 is the image generated by a fluorescence imager showing the products produced by the cleavage of circular M13 DNA using Cleavase® BN/thrombin.

Figure 75 is an SDS-PAGE gel showing the migration of purified Cleavase® BN nuclease, Pfu FEN-1, Pwo FEN-1 and Mja FEN-1.

Figure 76 is the image generated by a fluorescence imager showing the products 20 produced by the cleavage of the S-33 and 11-8-0 oligonucleotides by Cleavase® BN and the Mja FEN-1 nucleases.

Figure 77 is the image generated by a fluorescence imager showing the products produced by the incubation of an oligonucleotide either having or lacking a 3'-OH group with TdT.

*oooo Figure 78 is the image generated by a fluorescence imager showing the products produced the incubation of cleavage products with TdT.

Figure 79 is a photograph of a Universal GeneCombTM card showing the capture and detection of cleavage products on a nitrocellulose support.

Figure 80 is the image generated by a fluorescence imager showing the products 30 produced using the Cleavase® A/G and Pfu FEN-I nucleases and a fluorescein-labeled probe.

Figure 81 is the image generated by a fluorescence imager showing the products produced using the Cleavase® A/G and Pfu FEN-1 nucleases and a Cy3-labeled probe.

27 Figure 82 is the image generated by a fluorescence imager showing the products produced using the Cleavase® A/G and Pfu FEN-1 nucleases and a TET-labeled probe.

Figures 83A and 83B are images generated by a fluorescence imager showing the products produced using the Cleavase® A/G and Pfu FEN-1 nucleases and probes having or lacking a 5' positive charge; the gel shown in Fig. 83A was run in the standard direction and the gel shown in Fig. 84B was run in the reverse direction.

Figure 84 shows the structure of 3-nitropyrrole and Figure 85 shows the sequence of oligonucleotides 109, 61 and 67 (SEQ ID NOS:83, 61 and 62) annealed into a cleavage structure as well as the sequence of oligonucleotide 67 (SEQ ID NO:62) and a composite of SEQ ID NOS:84-88.

Figure 86A-C show images generated by a fluorescence imager showing the products produced in an InvaderTM-directed cleavage assay performed at various temperatures using a miniprobe which is either completely complementary to the target or contains a single mismatch with the target.

Figure 87 shows the sequence of oligonucleotides 166 (SEQ ID NO:93), 165 (SEQ ID NO:92), 161 (SEQ ID NO:89), 162 (SEQ ID NO:90) and 164 (SEQ ID NO:91) as well as a cleavage structure.

Figures 88 shows the image generated by a fluorescence imager showing the products produced in an InvaderTM-directed cleavage assay performed using ras gene sequences as the 20 target.

Figures 89A-C show the sequence of the S-60 hairpin (SEQ ID NO:40) and the oligonucleotide (SEQ ID NO:41) (shown annealed to the S-60 hairpin in B) and the image generated by a fluorescence imager showing the products produced by cleavage of the hairpin in the presence of various InvaderTM oligonucleotides.

25 Figure 90 shows the structure of various 3' end substituents.

Figure 91 is a composite graph showing the effect of probe concentration, temperature and a stacker oligonucleotide on the cleavage of miniprobes.

Figure 92 shows the sequence of the IT-2 oligonucleotide (SEQ ID NO:123; shown in a folded configuration) as well as the sequence of the IT-1 (SEQ ID NO:124) and IT-1A4 (SEQ ID NO:125) oligonucleotides.

Figure 93 shows the image generated by a fluorescence imager showing the products produced cleavage of the oligonucleotides shown in Figure 92 by Cleavase® A/G nuclease.

28 Figure 94 shows the image generated by a fluorescence imager which provides a comparison of the rates of cleavage by the Pfu FEN-1 and Mja FEN-l nucleases.

Fig,,e 95 shews he image ener a ai wcic udepicts the detection of RNA targets using a miniprobe and stacker oligonucleotides.

Figure 96A shows the image generated by a fluorescence imager comparing the products produced by cleavage of a mixture of the oligonucleotides shown in Figure 71 by either Pfu FEN-1 or Mja FEN-1 Figure 96B shows the image generated by a fluorescence imager comparing the products produced by cleavage of the oligonucleotides shown in Figure 30 by either Pfu FEN- 1 or Mja FEN-l Figure 97 shows a schematic diagram of the portions of the Pfu FEN-1 and Mja FEN- 1 proteins combined to create chimeric nucleases.

Figure 98A shows the image generated by a fluorescence imager comparing the products produced by cleavage of a mixture of the oligonucleotides shown in Figure 71 by Pfu FEN-1 Mja FEN-1 or the chimeric nucleases diagrammed in Figure 97.

Figure 98B shows the image generated by a fluorescence imager comparing the products produced by cleavage of the oligonucleotides shown in Figure 30 by Pfu FEN-1 Mja FEN-I or the chimeric nucleases diagrammed in Figure 97.

Figure 99 shows the image generated by a fluorescence imager comparing the products 20 produced by cleavage of folded cleavage structures by Pfu FEN-1 Mja FEN-1 or the chimeric nucleases diagrammed in Figure 97.

Figure 100A-J shows the results of various assays used to determine the activity of Cleavase BN under various conditions.

Figure 101A-B, D-F, and H-J show the results of various assays used to determine the 25 activity of TaqDN under various conditions.

Figure 102A-B, D-F, H-J show the results of various assays used to determine the activity of TthDN under various conditions.

Figure 103A-B, D-F, and H-J show the results of various assays used to determine the activity of Pfu FEN-1 under various conditions.

30 Figure 104A-J show the results of various assays used to determine the activity of Mja FEN-1 under various conditions.

Figure 105A-B, D-F, and H-J show the results of various assays used to determine the activity of Afu FEN-1 under various conditions.

29-

I

Figure 106A-E, and G-l show the results of various assays used to determine the activity of Mth FEN-I under various conditions.

Figure 107 shows the two substrates. Panel A shows the structure and sequence of the hairpin sulsijua[ (2-65-i)(SEQ ID NO:176), while Panel B shows the structure and sequence of the invader (IT) substrate (25-184-5)(SEQ ID NO:177).

DEFINITIONS

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence is complementary to the sequence Complementarity may be "partial." in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or. there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The term "homology" refers to a degree of identity. There may be partial homology or complete homology. A partially identical sequence is one that is less than 100% identical 20 to another sequence.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the 25 Tm of the formed hybrid, and the G:C ratio within the nucleic acids.

As used herein, the term is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm 81.5 0.41(% G when a nucleic acid is in aqueous solution at 1 M NaCI (see Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references include more 30 '1 sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.

As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required when it is desired that nucleic acids which are not completely complementary to one another be hybridized or annealed together.

The term "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired enzymatic activity is retained.

The term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product which displays modifications in sequence and or functional °oo. properties altered characteristics) when compared to the wild-type gene or gene product.

20 It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that ;°°they have altered characteristics when compared to the wild-type gene or gene product.

The term "recombinant DNA vector" as used herein refers to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism. DNA 25 sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, polyadenlyation signals and enhancers.

°o.ooi The term "LTR" as used herein refers to the long terminal repeat found at each end of a provirus the integrated form of a retrovirus). The LTR contains numerous regulatory 30 signals including transcriptional control elements, polyadenylation signals and sequences o r needed for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R and -31 The U3 region contains the enhancer and promoter elements. The U5 region contains the polyadenylation signals. The R (repeat) region separates the U3 and U5 regions and i ransci.bcd 3L quO %f thflI R I nIUI JL.WII UL LLII Ua b WIh 5 a Ud U3 e Sf t aL ViralA.

The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to nucleotides. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the end" if its 3' oxygen is not linked to a phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.

A first region along a nucleic acid strand is said to be upstream of another region if the 3' end of the first region is before the 5' end of the second region when moving along a strand 20 of nucleic acid in a 5' to 3' direction.

When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the "upstream" oligonucleotide and the latter the "downstream" oligonucleotide.

25 The term "primer" refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated.

An oligonucleotide "primer" may occur naturally, as in a purified restriction digest or may be produced synthetically.

A primer is selected to be "substantially" complementary to a strand of specific 30 sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being 32 substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby for a template primer complex for synthesis of the extension product of the primer.

"Hybridization" methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected; the detection of this sequence may be by either direct or indirect means). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the "hybridization" process by Marmur and Lane, Proc. Natl. Acad Sci. USA 46:453 (1960) and Doty et al., Proc. Natl.

Acad Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modem biology.

With regard to complementarity, it is important for some diagnostic applications to determine whether the hybridization represents complete or partial complementarity. For 15 example, where it is desired to detect simply the presence or absence of pathogen DNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan) it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions 0 can be selected where both partially complementary probes and completely complementary probes will hybridize. Other diagnostic applications, however, may require that the hybridization method distinguish between partial and complete complementarity. It may be of *i interest to detect genetic polymorphisms. For example, human hemoglobin is composed, in part, of four polypeptide chains. Two of these chains are identical chains of 141 amino acids (alpha chains) and two of these chains are identical chains of 146 amino acids (beta chains).

S°The gene encoding the beta chain is known to exhibit polymorphism. The normal allele encodes a beta chain having glutamic acid at the sixth position. The mutant allele encodes a beta chain having valine at the sixth position. This difference in amino acids has a profound (most profound when the individual is homozygous for the mutant allele) physiological impact known clinically as sickle cell anemia. It is well known that the genetic basis of the amino acid change involves a single base difference between the normal allele DNA sequence and the mutant allele DNA sequence.

The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association." Certain bases not 33 commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the melting temperature, or "Tm." The Tm of a particular nucleic acid duplex under specified conditions is the temperature at which on average half of the base pairs have disassociated.

The term "label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and 15 the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral.

•The term "cleavage structure" as used herein, refers to a structure which is formed by the interaction of a probe oligonucleotide and a target nucleic acid to form a duplex, the resulting structure being cleavable by a cleavage means, including but not limited to an enzyme. The cleavage structure is a substrate for specific cleavage by the cleavage means in contrast to a nucleic acid molecule which is a substrate for non-specific cleavage by agents such as phosphodiesterases which cleave nucleic acid molecules without regard to secondary structure no formation of a duplexed structure is required).

The term "folded cleavage structure" as used herein, refers to a region of a single- 25 stranded nucleic acid substrate containing secondary structure, the region being cleavable by an enzymatic cleavage means. The cleavage structure is a substrate for specific cleavage by S* the cleavage means in contrast to a nucleic acid molecule which is a substrate for non-specific cleavage by agents such as phosphodiesterases which cleave nucleic acid molecules without regard to secondary structure no folding of the substrate is required).

As used herein, the term "folded target" refers to a nucleic acid strand that contains at least one region of secondary structure at least one double stranded region and at least one single-stranded region within a single strand of the nucleic acid). A folded target may comprise regions of tertiary structure in addition to regions of secondary structure.

34 The term "cleavage means" as used herein refers to any means which is capable of cleaving a cleavage structure, including but not limited to enzymes. The cleavage means may include native DNAPs having 5' nuclease activity Taq DNA polymerase, E. coli DNA poiymerase 1) and, more specifically, modified DNAPs having 5' nuclease but lacking synthetic activity. The ability of 5' nucleases to cleave naturally occurring structures in nucleic acid templates (structure-specific cleavage) is useful to detect internal sequence differences in nucleic acids without prior knowledge of the specific sequence of the nucleic acid. In this manner, they are structure-specific enzymes. "Structure-specific nucleases" or "structure-specific enzymes" are enzymes which recognize specific secondary structures in a nucleic molecule and cleave these structures. The cleavage means of the invention cleave a nucleic acid molecule in response to the formation of cleavage structures; it is not necessary that the cleavage means cleave the cleavage structure at any particular location within the cleavage structure.

The cleavage means is not restricted to enzymes having solely 5' nuclease activity.

15 The cleavage means may include nuclease activity provided from a variety of sources e including the Cleavase® enzymes, the FEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNA polymerase and E. coli DNA polymerase I.

The term "thermostable" when used in reference to an enzyme, such as a 5' nuclease, indicates that the enzyme is functional or active can perform catalysis) at an elevated temperature, at about 55°C or higher.

The term "cleavage products" as used herein, refers to products generated by the reaction of a cleavage means with a cleavage structure the treatment of a cleavage structure with a cleavage means).

.The term "target nucleic acid" refers to a nucleic acid molecule which contains a 25 sequence which has at least partial complementarity with at least a probe oligonucleotide and may also have at least partial complementarity with an invader oligonucleotide. The target nucleic acid may comprise single- or double-stranded DNA or RNA.

°The term "probe oligonucleotide" refers to an oligonucleotide which interacts with a target nucleic acid to form a cleavage structure in the presence or absence of an invader oligonucleotide. When annealed to the target nucleic acid, the probe oligonucleotide and target form a cleavage structure and cleavage occurs within the probe oligonucleotide. In the presence of an invader oligonucleotide upstream of the probe oligonucleotide along the target 35 nucleic acid will shift the site of cleavage within the probe oligonucleotide (relative to the site of cleavage in the absence of the invader).

The term "non-target cleavage product" refers to a product of a cleavage reaction which is not derived from the target nucleic acid. As discussed above, in the methods of the present invention, cleavage of the cleavage structure occurs within the probe oligonucleotide.

The fragments of the probe oligonucleotide generated by this target nucleic acid-dependent cleavage are "non-target cleavage products." The term "invader oligonucleotide" refers to an oligonucleotide which contains sequences at its 3' end which are substantially the same as sequences located at the 5' end of a probe oligonucleotide; these regions will compete for hybridization to the same segment along a complementary target nucleic acid.

The term "substantially single-stranded" when used in reference to a nucleic acid substrate means that the substrate molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded substrate which exists as two strands of nucleic acid which are held together by inter-strand base pairing interactions.

The term "sequence variation" as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of this wild-type structural gene may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. A second mutant form of the structural gene may exist. This second mutant form is said to vary in sequence from both the wild-type gene and the first mutant form of the gene.

The term "liberating" as used herein refers to the release of a nucleic acid fragment from a larger nucleic acid fragment, such as an oligonucleotide, by the action of a 5' nuclease 25 such that the released fragment is no longer covalently attached to the remainder of the oligonucleotide.

The term as used herein refers to the Michaelis-Menten constant for an enzyme o* and is defined as the concentration of the specific substrate at which a given enzyme yields one-half its maximum velocity in an enzyme catalyzed reaction.

The term "nucleotide analog" as used herein refers to modified or non-naturally occurring nucleotides such as 7-deaza purines 7-deaza-dATP and 7-deaza-dGTP).

Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.

36- The term "polymorphic locus" is a locus present in a population which shows variation between members of the population the most common allele has a frequency of less than 0.95). In contrast, a "monomorphic locus" is a genetic locus at little or no variations seen between members of the population (generally taken to be a locus at which the most common allele exceeds a frequency of 0.95 in the gene pool of the population).

The term "microorganism" as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi, and ciliates.

The term "microbial gene sequences" refers to gene sequences derived from a microorganism.

The term "bacteria" refers to any bacterial species including eubacterial and archaebacterial species.

The term "virus" refers to obligate, ultramicroscopic, intracellular parasites incapable of autonomous replication replication requires the use of the host cell's machinery).

15 The term "multi-drug resistant" or multiple-drug resistant" refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.

The term "sample" in the present specification and claims is used in its broadest sense.

On the one hand it is meant to include a specimen or culture microbiological cultures).

On the other hand, it is meant to include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but ooe S' 25 not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.

Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing S"instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

-37- The term "source of target nucleic acid" refers to any sample which contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk.

lymph, sputum and semen.

An oligonucleotide is said to be present in "excess" relative to another oligonucleotide (or target nucleic acid sequence) if that oligonucleotide is present at a higher molar concentration that the other oligonucleotide (or target nucleic acid sequence). When an oligonucleotide such as a probe oligonucleotide is present in a cleavage reaction in excess relative to the concentration of the complementary target nucleic acid sequence, the reaction may be used to indicate the amount of the target nucleic acid present. Typically, when present in excess, the probe oligonucleotide will be present at least a 100-fold molar excess; typically at least 1 pmole of each probe oligonucleotide would be used when the target nucleic acid sequence was present at about 10 fmoles or less.

A sample "suspected of containing" a first and a second target nucleic acid may contain either. both or neither target nucleic acid molecule.

The term "charge-balanced" oligonucleotide refers to an oligonucleotide (the input oligonucleotide in a reaction) which has been modified such that the modified oligonucleotide bears a charge, such that when the modified oligonucleotide is either cleaved shortened) or elongated, a resulting product bears a charge different from the input oligonucleotide (the "charge-unbalanced" oligonucleotide) thereby permitting separation of the input and reacted oligonucleotides on the basis of charge. The term "charge-balanced" does not imply that the modified or balanced oligonucleotide has a net neutral charge (although this can be the case).

Charge-balancing refers to the design and modification of an oligonucleotide such that a specific reaction product generated from this input oligonucleotide can be separated on the basis of charge from the input oligonucleotide.

For example, in an invader-directed cleavage assay in which the probe oligonucleotide S: bears the sequence: 5'-TTCTTTTCACCAGCGAGACGGG-3' SEQ ID NO:61 without the modified bases) and cleavage of the probe occurs between the second and third residues, one possible charge-balanced version of this oligonucleotide would be: 5'-Cy3-AminoT- Amino-TCTTTTCACCAGCGAGAC GGG-3'. This modified oligonucleotide bears a net negative charge. After cleavage, the following oligonucleotides are generated: 5'-Cy3- AminoT-Amino-T-3'and 5'-CTTTTCACCAGCGAGACGGG-3' (residues 3-22of SEQ ID NO:61). 5'-Cy3-AminoT-Amino-T-3'bears a detectable moiety (the positively-charged Cy3 -38 dye) and two amino-modified bases. The amino-modified bases and the Cy3 dye contribute positive charges in excess of the negative charges contributed by the phosphate groups and thus the 5'-Cy3-AminoT-Amino-T-3'oligonucleotide has a net positive charge. The oter, longer cleavage fragment, like the input probe, bears a net negative charge. Because the 5'-Cy3-AminoT-Amino-T-3'fragment is separable on the basis of charge from the input probe (the charge-balanced oligonucleotide), it is referred to as a charge-unbalanced oligonucleotide.

The longer cleavage product cannot be separated on the basis of charge from the input oligonucleotide as both oligonucleotides bear a net negative charge; thus, the longer cleavage product is not a charge-unbalanced oligonucleotide.

The term "net neutral charge" when used in reference to an oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present R-NH 3 groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction conditions is essentially zero. An oligonucleotide having a net neutral charge would not migrate in an electrical field.

15 The term "net positive charge" when used in reference to an oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present R-NH 3 groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction conditions is +1 or greater. An oligonucleotide having a net positive charge would migrate toward the negative electrode in an electrical field.

The term "net negative charge" when used in reference to an oligonucleotide, including modified oligonucleotides, indicates that the sum of the charges present R-NH 3 groups on thymidines, the N3 nitrogen of cytosine, presence or absence or phosphate groups, etc.) under the desired reaction conditions is -1 or lower. An oligonucleotide having a net negative charge would migrate toward the positive electrode in an electrical field.

The term "polymerization means" refers to any agent capable of facilitating the addition of nucleoside triphosphates to an oligonucleotide. Preferred polymerization means comprise DNA polymerases.

The term "ligation means" refers to any agent capable of facilitating the ligation the formation of a phosphodiester bond between a 3'-OH and a 5'-P located at the termini of two strands of nucleic acid). Preferred ligation means comprise DNA ligases and RNA ligases.

39 The term "reactant" is used herein in its broadest sense. The reactant can comprise an enzymatic reactant, a chemical reactant or ultraviolet light (ultraviolet light, particularly short wavelength ultraviolet light is known to break oligonucleotide chains). Any agent capable of reacting with an oligonucleotide to either shorten cleave) or elongate the oligonucleotide is encompassed within the term "reactant." The term "adduct" is used herein in its broadest sense to indicate any compound or element which can be added to an oligonucleotide. An adduct may be charged (positively or negatively) or may be charge neutral. An adduct may be added to the oligonucleotide via covalent or non-covalent linkages. Examples of adducts, include but are not limited to indodicarbocyanine dye amidites, amino-substituted nucleotides, ethidium bromide, ethidium homodimer, (1,3-propanediamino)propidium, (diethylenetriamino)propidium, thiazole orange, (N-N'-tetramethyl-l,3-propanediamino)propyl thiazole orange, (N-N'-tetramethyl-1,2ethanediamino)propyl thiazole orange, thiazole orange-thiazole orange homodimer (TOTO), thiazole orange-thiazole blue heterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED thiazole orange-ethidium heterodimer 2 (TOED2) and fluorescein-ethidium heterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

Where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a "region of overlap" exists along the target nucleic acid. The degree of overlap will vary depending upon the nature of the complementarity (see. region in Figs. 29 and 67 and the accompanying discussions).

As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, recombinant Cleavase® nucleases are expressed in bacterial host cells and the nucleases are purified by the removal of host cell proteins; the 25 percent of these recombinant nucleases is thereby increased in the sample.

The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed from a recombinant DNA molecule.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

"Nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or svnthetir nrigin whirh may be singnle nr rndcb.loe srded, and Ie s iS.

strand. Similarly, "amino acid sequence" as used herein refers to peptide or protein sequence.

"Peptide nucleic acid" as used herein refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid [Nielsen PE el al. (1993) Anticancer Drug Des. 8:53-63].

As used herein, the terms "purified" or "substantially purified" refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably free from other components with which they are naturally associated. An "isolated polynucleotide" or "isolated oligonucleotide" is therefore a substantially purified 15 polynucleotide.

An isolated oligonucleotide (or polynucleotide) encoding a Pyrococcus woesei (Pwo) -FEN-1 endonuclease having a region capable of hybridizing to SEQ ID NO: 116 is an oligonucleotide containing sequences encoding at least the amino-terminal portion of Pwo FEN- endonuclease. An isolated oligonucleotide (or polynucleotide) encoding a Pwo FEN-1 endonuclease having a region capable of hybridizing to SEQ ID NO:117 is an oligonucleotide ~containing sequences encoding at least the carboxy-terminal portion of Pwo FEN-I endonuclease. An isolated oligonucleotide (or polynucleotide) encoding a Pwo FEN-1 endonuclease having a region capable of hybridizing to SEQ ID NOS:118 and 119 is an oligonucleotide containing sequences encoding at least portions of Pwo FEN-I endonuclease protein located internal to either the amino or carboxy-termini of the Pwo FEN-1 endonuclease protein.

As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest Cleavase® BN/thrombin nuclease and portions or fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-Cleavase® BN/thrombin nuclease protein). The fusion partner may enhance solubility of recombinant chimeric protein the Cleavase® BN/thrombin nuclease) as expressed in a host cell. may provide an affinity tag a his-tag) to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. If desired, the fusion protein -41 may be removed from the protein of interest Cleavase® BN/thrombin nuclease or fragments thereof) by a variety of enzymatic or chemical means known to the art.

As used herein, the terms "chimeric protein" and "chimerical protein" refer to a single protein iiiulcuuie ihat comprises amino acid sequences portions derived from two or more parent proteins. These parent molecules may be from similar proteins from genetically distinct origins, different proteins from a single organism, or different proteins from different organisms. By way of example but not by way of limitation, the a chimeric structure-specific nuclease of the present invention may contain a mixture of amino acid sequences that have been derived from FEN-1 genes from two or more of the organisms having such genes, combined to form a non-naturally occurring nuclease. The term "chimerical" as used herein is not intended to convey any particular proportion of contribution from the naturally occurring genes, nor limit the manner in which the portions are combined. Any chimeric structurespecific nuclease constructs having cleavage activity as determined by the testing methods described herein are improved cleavage agents within the scope of the present invention.

DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for treating nucleic acid.

and in particular, methods and compositions for detection and characterization of nucleic acid sequences and sequence changes.

The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. In particular, the present invention relates to a cleaving enzyme having 5' nuclease activity without interfering nucleic acid synthetic ability.

This invention provides 5' nucleases derived from thermostable DNA polymerases which exhibit altered DNA synthetic activity from that of native thermostable DNA 25 polymerases. The 5' nuclease activity of the polymerase is retained while the synthetic activity is reduced or absent. Such 5' nucleases are capable of catalyzing the structure- *specific cleavage of nucleic acids in the absence of interfering synthetic activity. The lack of synthetic activity during a cleavage reaction results in nucleic acid cleavage products of uniform size.

The novel properties of the nucleases of the invention form the basis of a method of detecting specific nucleic acid sequences. This method relies upon the amplification of the detection molecule rather than upon the amplification of the target sequence itself as do existing methods of detecting specific target sequences.

-42 .j DNA polymerases (DNAPs), such as those isolated from E. coli or from thermophilic bacteria of the genus Thermus, are enzymes that synthesize new DNA strands. Several of the known DNAPs contain associated nuclease activities in addition to the synthetic activity of the enzyme.

Some DNAPs are known to remove nucleotides from the 5' and 3' ends of DNA chains (Kornberg, DNA Replication, W.H. Freeman and Co., San Francisco, pp. 127-139 [1980]). These nuclease activities are usually referred to as 5' exonuclease and 3' exonuclease activities, respectively. For example, the 5' exonuclease activity located in the N-terminal domain of several DNAPs participates in the removal of RNA primers during lagging strand synthesis during DNA replication and the removal of damaged nucleotides during repair. Some DNAPs, such as the E. coli DNA polymerase (DNAPEcl), also have a 3' exonuclease activity responsible for proof-reading during DNA synthesis (Kornberg.

supra).

A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase (DNAPTaq), has a 5' exonuclease activity, but lacks a functional 3' exonucleolytic domain (Tindall and Kunkell, Biochem., 27:6008 [1988]). Derivatives of DNAPEcl and DNAPTaq, respectively called the Klenow and Stoffel fragments, lack 5' exonuclease domains as a result of enzymatic or genetic manipulations (Brutlag et al., Biochem. Biophys. Res. Commun.. 37:982 [1969]; Erlich et al., Science 252:1643 [1991]; Setlow and Kornberg, J. Biol. Chem.. 247:232 [1972]).

The 5' exonuclease activity of DNAPTaq was reported to require concurrent synthesis (Gelfand, PCR Technology Principles and Applications for DNA Amplification, H.A. Erlich, Stockton Press, New York, p. 19 [1989]). Although mononucleotides predominate among the digestion products of the 5' exonucleases of DNAPTaq and DNAPEcl, short 25 oligonucleotides (5 12 nucleotides) can also be observed implying that these so-called exonucleases can function endonucleolytically (Setlow, supra; Holland et al., Proc. Natl.

Acad. Sci. USA 88:7276 [1991]).

In WO 92/06200, Gelfand el al. show that the preferred substrate of the exonuclease activity of the thermostable DNA polymerases is displaced single-stranded DNA.

Hydrolysis of the phosphodiester bond occurs between the displaced single-stranded DNA and the double-helical DNA with the preferred exonuclease cleavage site being a phosphodiester bond in the double helical region. Thus, the 5' exonuclease activity usually associated with DNAPs is a structure-dependent single-stranded endonuclease and is more properly referred to 43 as a 5' nuclease. Exonucleases are enzymes which cleave nucleotide molecules from the ends of the nucleic acid molecule. Endonucleases, on the other hand, are enzymes which cleave the nucleic acid molecule at internal rather than terminal sites. The nuclease activity associated with some thermostable DNA polymerases cleaves endonucleolytically but this cleavage requires contact with the 5' end of the molecule being cleaved. Therefore, these nucleases are referred to as 5' nucleases.

When a 5' nuclease activity is associated with a eubacterial Type A DNA polymerase, it is found in the one-third N-terminal region of the protein as an independent functional domain. The C-terminal two-thirds of the molecule constitute the polymerization domain which is responsible for the synthesis of DNA. Some Type A DNA polymerases also have a 3' exonuclease activity associated with the two-third C-terminal region of the molecule.

The 5' exonuclease activity and the polymerization activity of DNAPs have been separated by proteolytic cleavage or genetic manipulation of the polymerase molecule. To date thermostable DNAPs have been modified to remove or reduce the amount of 5' nuclease activity while leaving the polymerase activity intact.

The Klenow or large proteolytic cleavage fragment of DNAPEcl contains the polymerase and 3' exonuclease activity but lacks the 5' nuclease activity. The Stoffel fragment of DNAPTaq (DNAPStf) lacks the 5' nuclease activity due to a genetic manipulation which deleted the N-terminal 289 amino acids of the polymerase molecule (Erlich et al., Science 252:1643 [1991]). WO 92/06200 describes a thermostable DNAP with an altered level of 5' to 3' exonuclease. U.S. Patent No. 5,108,892 describes a Thermus aquaticus DNAP without a 5' to 3' exonuclease. However, the art of molecular biology lacks a thermostable DNA polymerase with a lessened amount of synthetic activity.

The present invention provides 5' nucleases derived from thermostable Type A DNA 25 polymerases that retain 5' nuclease activity but have reduced or absent synthetic activity. The ability to uncouple the synthetic activity of the enzyme from the 5' nuclease activity proves f* that the 5' nuclease activity does not require concurrent DNA synthesis as was previously reported (Gelfand, PCR Technology, supra).

The description of the invention is divided into: I. Detection of Specific Nucleic Acid Sequences Using 5' Nucleases; II. Generation of 5' Nucleases Derived From Thermostable DNA Polymerases; III. Detection of Specific Nucleic Acid Sequences Using 5' Nucleases in an Invader-Directed Cleavage Assay; IV. A Comparison Of Invasive Cleavage And Primer-Directed Cleavage; V. Fractionation Of Specific Nucleic Acids By Selective Charge -44 Reversal; VI. InvaderrM-Directed Cleavage Using Miniprobes And Mid-Range Probes; VII.

Signal Enhancement By Tailing Of Reaction Products In The InvaderTM-Directed Cleavage Assay VIII. Improved Enzymes For .Is In In nvaderTM-Directed C!e(Vage Reactions; and IX.

Improved Enzymes For Use in The CFLP® Method.

I. Detection Of Specific Nucleic Acid Sequences Using 5' Nucleases The 5' nucleases of the invention form the basis of a novel detection assay for the identification of specific nucleic acid sequences. This detection system identifies the presence of specific nucleic acid sequences by requiring the annealing of two oligonucleotide probes to two portions of the target sequence. As used herein, the term "target sequence" or "target nucleic acid sequence" refers to a specific nucleic acid sequence within a polynucleotide sequence, such as genomic DNA or RNA, which is to be either detected or cleaved or both.

Fig. IA provides a schematic of one embodiment of the detection method of the present invention. The target sequence is recognized by two distinct oligonucleotides in the 15 triggering or trigger reaction. It is preferred that one of these oligonucleotides is provided on a solid support. The other can be provided free. In Fig. 1A the free oligonucleotide is indicated as a "primer" and the other oligonucleotide is shown attached to a bead designated as type 1. The target nucleic acid aligns the two oligonucleotides for specific cleavage of the 5' arm (of the oligonucleotide on bead 1) by the DNAPs of the present invention (not shown in Fig. 1A).

The site of cleavage (indicated by a large solid arrowhead) is controlled by the distance between the 3' end of the "primer" and the downstream fork of the oligonucleotide on bead 1. The latter is designed with an uncleavable region (indicated by the striping). In S* °this manner neither oligonucleotide is subject to cleavage when misaligned or when unattached to target nucleic acid.

Successful cleavage releases a single copy of what is referred to as the alpha signal oligonucleotide. This oligonucleotide may contain a detectable moiety fluorescein). On the other hand, it may be unlabelled.

In one embodiment of the detection method, two more oligonucleotides are provided on solid supports. The oligonucleotide shown in Fig. 1A on bead 2 has a region that is complementary to the alpha signal oligonucleotide (indicated as alpha prime) allowing for hybridization. This structure can be cleaved by the DNAPs of the present invention to release the beta signal oligonucleotide. The beta signal oligonucleotide can then hybridize to type 3 beads having an oligonucleotide with a complementary region (indicated as beta prime).

Again, this structure can be cleaved by the DNAPs of the present invention to release a new alpha oligonucleotide.

At this point, the amplification has been linear. To increase the power of the method, it is desired that the alpha signal oligonucleotide hybridized to bead type 2 be liberated after release of the beta oligonucleotide so that it may go on to hybridize with other oligonucleotides on type 2 beads. Similarly, after release of an alpha oligonucleotide from type 3 beads, it is desired that the beta oligonucleotide be liberated.

The liberation of "captured" signal oligonucleotides can be achieved in a number of ways. First, it has been found that the DNAPs of the present invention have a true exonuclease capable of "nibbling" the 5' end of the alpha (and beta) prime oligonucleotide (discussed below in more detail). Thus, under appropriate conditions, the hybridization is destabilized by nibbling of the DNAP. Second, the alpha alpha prime (as well as the beta beta prime) complex can be destabilized by heat thermal cycling).

With the liberation of signal oligonucleotides by such techniques. each cleavage results in a doubling of the number of signal oligonucleotides. In this manner, detectable signal can *g quickly be achieved.

Fig. 1B provides a schematic of a second embodiment of the detection method of the present invention. Again, the target sequence is recognized by two distinct oligonucleotides in the triggering or trigger reaction and the target nucleic acid aligns the two oligonucleotides for specific cleavage of the 5' arm by the DNAPs of the present invention (not shown in Fig.

S1B). The first oligonucleotide is completely complementary to a portion of the target sequence. The second oligonucleotide is partially complementary to the target sequence; the 3' end of the second oligonucleotide is fully complementary to the target sequence while the 25 5' end is non-complementary and forms a single-stranded arm. The non-complementary end of the second oligonucleotide may be a generic sequence which can be used with a set of standard hairpin structures (described below). The detection of different target sequences would require unique portions of two oligonucleotides: the entire first oligonucleotide and the 3' end of the second oligonucleotide. The 5' arm of the second oligonucleotide can be invariant or generic in sequence.

The annealing of the first and second oligonucleotides near one another along the target sequence forms a forked cleavage structure which is a substrate for the 5' nuclease of -46- DNA polymerases. The approximate location of the cleavage site is again indicated by the large solid arrowhead in Fig. 1B.

The 5' nucleases of the invention are capable of cleaving this stmrctture bhut rpe no* capable of polymerizing the extension of the 3' end of the first oligonucleotide. The lack of polymerization activity is advantageous as extension of the first oligonucleotide results in displacement of the annealed region of the second oligonucleotide and results in moving the site of cleavage along the second oligonucleotide. If polymerization is allowed to occur to any significant amount, multiple lengths of cleavage product will be generated. A single cleavage product of uniform length is desirable as this cleavage product initiates the detection reaction.

The trigger reaction may be run under conditions that allow for thermocycling.

Thermocycling of the reaction allows for a logarithmic increase in the amount of the trigger oligonucleotide released in the reaction.

The second part of the detection method allows the annealing of the fragment of the 15 second oligonucleotide liberated by the cleavage of the first cleavage structure formed in the triggering reaction (called the third or trigger oligonucleotide) to a first hairpin structure.

This first hairpin structure has a single-stranded 5' arm and a single-stranded 3' arm. The third oligonucleotide triggers the cleavage of this first hairpin structure by annealing to the 3' arm of the hairpin thereby forming a substrate for cleavage by the 5' nuclease of the present invention. The cleavage of this first hairpin structure generates two reaction products: 1) the cleaved 5' arm of the hairpin called the fourth oligonucleotide, and 2) the cleaved hairpin structure which now lacks the 5' arm and is smaller in size than the uncleaved hairpin. This cleaved first hairpin may be used as a detection molecule to indicate that cleavage directed by the trigger or third oligonucleotide occurred. Thus, this indicates that the first two 25 oligonucleotides found and annealed to the target sequence thereby indicating the presence of the target sequence in the sample.

The detection products are amplified by having the fourth oligonucleotide anneal to a second hairpin structure. This hairpin structure has a 5' single-stranded arm and a 3' singlestranded arm. The fourth oligonucleotide generated by cleavage of the first hairpin structure anneals to the 3' arm of the second hairpin structure thereby creating a third cleavage structure recognized by the 5' nuclease. The cleavage of this second hairpin structure also generates two reaction products: 1) the cleaved 5' arm of the hairpin called the fifth oligonucleotide which is similar or identical in sequence to the third nucleotide, and 2) the 47 cleaved second hairpin structure which now lacks the 5' arm and is smaller in size than the uncleaved hairpin. This cleaved second hairpin may be as a detection molecule and amplifies the signal generated by the cleavage of the first hairpin structure. Simultaneously with the an..aing of e forth oiigonucleotide, the third oligonucleotide is dissociated from the cleaved first hairpin molecule so that it is free to anneal to a new copy of the first hairpin structure. The disassociation of the oligonucleotides from the hairpin structures may be accomplished by heating or other means suitable to disrupt base-pairing interactions.

Further amplification of the detection signal is achieved by annealing the fifth oligonucleotide (similar or identical in sequence to the third oligonucleotide) to another molecule of the first hairpin structure. Cleavage is then performed and the oligonucleotide that is liberated then is annealed to another molecule of the second hairpin structure.

Successive rounds of annealing and cleavage of the first and second hairpin structures.

provided in excess. are performed to generate a sufficient amount of cleaved hairpin products to be detected. The temperature of the detection reaction is cycled just below and just above the annealing temperature for the oligonucleotides used to direct cleavage of the hairpin Sstructures, generally about 55°C to 70 0 C. The number of cleavages will double in each cycle •until the amount of hairpin structures remaining is below the K, for the hairpin structures.

This point is reached when the hairpin structures are substantially used up. When the detection reaction is to be used in a quantitative manner, the cycling reactions are stopped before the accumulation of the cleaved hairpin detection products reach a plateau.

Detection of the cleaved hairpin structures may be achieved in several ways. In one embodiment detection is achieved by separation on agarose or polyacrylamide gels followed by staining with ethidium bromide. In another embodiment, detection is achieved by separation of the cleaved and uncleaved hairpin structures on a gel followed by 25 autoradiography when the hairpin structures are first labeled with a radioactive probe and separation on chromatography columns using HPLC or FPLC followed by detection of the differently sized fragments by absorption at OD 26 0 Other means of detection include detection of changes in fluorescence polarization when the single-stranded 5' arm is released by cleavage, the increase in fluorescence of an intercalating fluorescent indicator as the amount of primers annealed to 3' arms of the hairpin structures increases. The formation of increasing amounts of duplex DNA (between the primer and the 3' arm of the hairpin) occurs if successive rounds of cleavage occur.

-48- The hairpin structures may be attached to a solid support, such as an agarose. styrene or magnetic bead, via the 3' end of the hairpin. A spacer molecule may be placed between the 3' end of the hairDin and the bead. if so desired The drl.ntenP f n i thai-:pii: structures to a solid support is that this prevents the hybridization of the two hairpin structures to one another over regions which are complementary. If the hairpin structures anneal to one another, this would reduce the amount of hairpins available for hybridization to the primers released during the cleavage reactions. If the hairpin structures are attached to a solid support, then additional methods of detection of the products of the cleavage reaction may be employed. These methods include, but are not limited to, the measurement of the released single-stranded 5' arm when the 5' arm contains a label at the 5' terminus. This label may be radioactive, fluorescent, biotinylated, etc. If the hairpin structure is not cleaved, the 5' label will remain attached to the solid support. If cleavage occurs, the 5' label will be released from the solid support.

The 3' end of the hairpin molecule may be blocked through the use of dideoxynucleotides. A 3' terminus containing a dideoxynucleotide is unavailable to participate in reactions with certain DNA modifying enzymes, such as terminal transferase.

Cleavage of the hairpin having a 3' terminal dideoxynucleotide generates a new, unblocked 3' terminus at the site of cleavage. This new 3' end has a free hydroxyl group which can interact with terminal transferase thus providing another means of detecting the cleavage products.

i The hairpin structures are designed so that their self-complementary regions are very short (generally in the range of 3-8 base pairs). Thus, the hairpin structures are not stable at the high temperatures at which this reaction is performed (generally in the range of 50-75°C) unless the hairpin is stabilized by the presence of the annealed oligonucleotide on the 3' arm 25 of the hairpin. This instability prevents the polymerase from cleaving the hairpin structure in the absence of an associated primer thereby preventing false positive results due to nonoligonucleotide directed cleavage.

As discussed above, the use of the 5' nucleases of the invention which have reduced polymerization activity is advantageous in this method of detecting specific nucleic acid sequences. Significant amounts of polymerization during the cleavage reaction would cause shifting of the site of cleavage in unpredictable ways resulting in the production of a series of cleaved hairpin structures of various sizes rather than a single easily quantifiable product.

Additionally, the primers used in one round of cleavage could, if elongated, become unusable -49for the next cycle, by either forming an incorrect structure or by being too long to melt off under moderate temperature cycling conditions. In a pristine system lacking the presence of dNTPs), one could use the unmodified polymerase, but the presence of nucleotides (dNTPs) can decrease the per cycle efficiency enough to give a false negative result. When a crude extract (genomic DNA preparations, crude cell lysates, etc.) is employed or where a sample of DNA from a PCR reaction, or any other sample that might be contaminated with dNTPs, the 5' nucleases of the present invention that were derived from thermostable polymerases are particularly useful.

II. Generation Of 5' Nucleases From Thermostable DNA Polymerases The genes encoding Type A DNA polymerases share about 85% homology to each other on the DNA sequence level. Preferred examples of thermostable polymerases include those isolated from Thermus aquaticus, Thermus flavus, and Thermus thermophilus. However, other thermostable Type A polymerases which have 5' nuclease activity are also suitable.

Figs. 2 and 3 compare the nucleotide and amino acid sequences of the three above mentioned polymerases. In Figs. 2 and 3, the consensus or majority sequence derived from a comparison of the nucleotide (Fig. 2) or amino acid (Fig. 3) sequence of the three thermostable DNA polymerases is shown on the top line. A dot appears in the sequences of each of these three polymerases whenever an amino acid residue in a given sequence is identical to that contained in the consensus amino acid sequence. Dashes are used to introduce gaps in order to maximize alignment between the displayed sequences. When no consensus nucleotide or **amino acid is present at a given position, an is placed in the consensus sequence. SEQ ID NOS:1-3 display the nucleotide sequences and SEQ ID NOS:4-6 display the amino acid sequences of the three wild-type polymerases. SEQ ID NO:1 corresponds to the nucleic acid i 25 sequence of the wild type Thermus aquaticus DNA polymerase gene isolated from the YT-I strain (Lawyer et al., J. Biol. Chem., 264:6427 [1989]). SEQ ID NO:2 corresponds to the S* nucleic acid sequence of the wild type Thermusflavus DNA polymerase gene (Akhmetzjanov and Vakhitov, Nucl. Acids Res., 20:5839 [1992]). SEQ ID NO:3 corresponds to the nucleic acid sequence of the wild type Thermus thermophilus DNA polymerase gene (Gelfand et al., WO 91/09950 [1991]). SEQ ID NOS:7-8 depict the consensus nucleotide and amino acid sequences, respectively for the above three DNAPs (also shown on the top row in Figs. 2 and 3).

i The 5' nucleases of the invention derived from thermostable polymerases have reduced synthetic ability, but retain substantially the same 5' exonuclease activity as the native DNA polymerase. The term "substantially the same 5' nc!ease activity" a used herein means that the 5' nuclease activity of the modified enzyme retains the ability to function as a structuredependent single-stranded endonuclease but not necessarily at the same rate of cleavage as compared to the unmodified enzyme. Type A DNA polymerases may also be modified so as to produce an enzyme which has increases 5' nuclease activity while having a reduced level of synthetic activity. Modified enzymes having reduced synthetic activity and increased nuclease activity are also envisioned by the present invention.

By the term "reduced synthetic activity" as used herein it is meant that the modified enzyme has less than the level of synthetic activity found in the unmodified or "native" enzyme. The modified enzyme may have no synthetic activity remaining or may have that level of synthetic activity that will not interfere with the use of the modified enzyme in the detection assay described below. The 5' nucleases of the present invention are advantageous 15 in situations where the cleavage activity of the polymerase is desired, but the synthetic ability is not (such as in the detection assay of the invention).

As noted above, it is not intended that the invention be limited by the nature of the alteration necessary to render the polymerase synthesis deficient. The present invention contemplates a variety of methods, including but not limited to: 1) proteolysis; 2) recombinant constructs (including mutants); and 3) physical and/or chemical modification *i and/or inhibition.

1. Proteolysis Thermostable DNA polymerases having a reduced level of synthetic activity are 25 produced by physically cleaving the unmodified enzyme with proteolytic enzymes to produce fragments of the enzyme that are deficient in synthetic activity but retain 5' nuclease activity.

Following proteolytic digestion, the resulting fragments are separated by standard chromatographic techniques and assayed for the ability to synthesize DNA and to act as a nuclease. The assays to determine synthetic activity and 5' nuclease activity are described below.

-51- 2. Recombinant Constructs The examples below describe a preferred method for creating a construct encoding a nuclease derived from a thermostable DNA polymerase. As the Type A DNA polymerases are similar in DNA seqenen the cl nin g strategics y fo he Thermus aquaicus and.

cg's Cllr.uycz lut iIX inermus aqualicus and T. flavus polymerases are applicable to other thermostable Type A polymerases. In general, a thermostable DNA polymerase is cloned by isolating genomic DNA using molecular biological methods from a bacteria containing a thermostable Type A DNA polymerase. This genomic DNA is exposed to primers which are capable of amplifying the polymerase gene by

PCR.

This amplified polymerase sequence is then subjected to standard deletion processes to delete the polymerase portion of the gene. Suitable deletion processes are described below in the examples.

The example below discusses the strategy used to determine which portions of the DNAPTaq polymerase domain could be removed without eliminating the 5' nuclease activity.

Deletion of amino acids from the protein can be done either by deletion of the encoding genetic material, or by introduction of a translational stop codon by mutation or frame shift.

In addition, proteolytic treatment of the protein molecule can be performed to remove segments of the protein.

In the examples below, specific alterations of the Taq gene were: a deletion between 20 nucleotides 1601 and 2502 (the end of the coding region), a 4 nucleotide insertion at position 2043, and deletions between nucleotides 1614 and 1848 and between nucleotides 875 and 1778 (numbering is as in SEQ ID NO:1). These modified sequences are described below in the examples and at SEQ ID NOS:9-12.

Those skilled in the art understand that single base pair changes can be innocuous in terms of enzyme structure and function. Similarly, small additions and deletions can be present without substantially changing the exonuclease or polymerase function of these enzymes.

Other deletions are also suitable to create the 5' nucleases of the present invention. It is preferable that the deletion decrease the polymerase activity of the 5' nucleases to a level at which synthetic activity will not interfere with the use of the 5' nuclease in the detection assay of the invention. Most preferably, the synthetic ability is absent. Modified polymerases are tested for the presence of synthetic and 5' nuclease activity as in assays described below.

Thoughtful consideration of these assays allows for the screening of candidate enzymes whose 52 structure is heretofore as yet unknown. In other words, construct can be evaluated according to the protocol described below to determine whether it is a member of the genus of 5' nucleases of the present invention as defined functionally, rather than structurally.

in the example below, the PCR product of the amplified Thermus aquaticus genomic DNA did not have the identical nucleotide structure of the native genomic DNA and did not have the same synthetic ability of the original clone. Base pair changes which result due to the infidelity of DNAPTaq during PCR amplification of a polymerase gene are also a method by which the synthetic ability of a polymerase gene may be inactivated. The examples below and Figs. 4A and 5A indicate regions in the native Thermus aquaticus and flavus DNA polymerases likely to be important for synthetic ability. There are other base pair changes and substitutions that will likely also inactivate the polymerase.

It is not necessary, however, that one start out the process of producing a 5' nuclease from a DNA polymerase with such a mutated amplified product. This is the method by which the examples below were performed to generate the synthesis-deficient DNAPTaq mutants, but it is understood by those skilled in the art that a wild-type DNA polymerase sequence may be used as the starting material for the introduction of deletions, insertion and substitutions to produce a 5' nuclease. For example, to generate the synthesis-deficient DNAPTfl mutant, the primers listed in SEQ ID NOS:13-14 were used to amplify the wild type DNA polymerase gene from Thermusflavus strain AT-62. The amplified polymerase 20 gene was then subjected to restriction enzyme digestion to delete a large portion of the domain encoding the synthetic activity.

The present invention contemplates that the nucleic acid construct of the present invention be capable of expression in a suitable host. Those in the art know methods for attaching various promoters and 3' sequences to a gene structure to achieve efficient expression. The examples below disclose two suitable vectors and six suitable vector constructs. Of course, there are other promoter/vector combinations that would be suitable. It is not necessary that a host organism be used-for the expression of the nucleic acid constructs of the invention. For example, expression of the protein encoded by a nucleic acid construct may be achieved through the use of a cell-free in vitro transcription/translation system. An 30 example of such a cell-free system is the commercially available TnTr M Coupled Reticulocyte Lysate System (Promega Corporation, Madison, WI).

o o 53 Once a suitable nucleic acid construct has been made, the 5' nuclease may be produced from the construct. The examples below and standard molecular biological teachings enable one to manipulate the construct by different suitable methods.

Once thc 5' nuclease lias been expressed, the polymerase is tested for both synthetic and nuclease activity as described below.

3. Physical And/Or Chemical Modification And/Or Inhibition The synthetic activity of a thermostable DNA polymerase may be reduced by chemical and/or physical means. In one embodiment, the cleavage reaction catalyzed by the nuclease activity of the polymerase is run under conditions which preferentially inhibit the synthetic activity of the polymerase. The level of synthetic activity need only be reduced to that level of activity which does not interfere with cleavage reactions requiring no significant synthetic activity.

As shown in the examples below, concentrations of Mg" greater than 5 mM inhibit the polymerization activity of the native DNAPTaq. The ability of the 5' nuclease to function under conditions where synthetic activity is inhibited is tested by running the assays for synthetic and 5' nuclease activity, described below, in the presence of a range of Mg" concentrations (5 to 10 mM). The effect of a given concentration of Mg*' is determined by quantitation of the amount of synthesis and cleavage in the test reaction as compared to the standard reaction for each assay.

The inhibitory effect of other ions, polyamines, denaturants, such as urea. formamide, dimethylsulfoxide, glycerol and non-ionic detergents (Triton X-100 and Tween-20). nucleic acid binding chemicals such as, actinomycin D, ethidium bromide and psoralens, are tested by their addition to the standard reaction buffers for the synthesis and 5' nuclease assays. Those compounds having a preferential inhibitory effect on the synthetic activity of a thermostable polymerase are then used to create reaction conditions under which 5' nuclease activity (cleavage) is retained while synthetic activity is reduced or eliminated.

Physical means may be used to preferentially inhibit the synthetic activity of a polymerase. For example, the synthetic activity of thermostable polymerases is destroyed by 30 exposure of the polymerase to extreme heat (typically 96 to 100 0 C) for extended periods of time (greater than or equal to 20 minutes). While these are minor differences with respect to the specific heat tolerance for each of the enzymes, these are readily determined. Polymerases 54 are treated with heat for various periods of time and the effect of the heat treatment upon the synthetic and 5' nuclease activities is determined.

III. Detection Of Specific Nucleic Acid Sequences Using 5' Nucleases In An Invader- Directed Cleavage Assay The present invention provides means for forming a nucleic acid cleavage structure which is dependent upon the presence of a target nucleic acid and cleaving the nucleic acid cleavage structure so as to release distinctive cleavage products. 5' nuclease activity is used to cleave the target-dependent cleavage structure and the resulting cleavage products are indicative of the presence of specific target nucleic acid sequences in the sample.

The present invention further provides assays in which the target nucleic acid is reused or recycled during multiple rounds of hybridization with oligonucleotide probes and cleavage without the need to use temperature cycling for periodic denaturation of target nucleic acid strands) or nucleic acid synthesis for the displacement of target nucleic acid strands). Through the interaction of the cleavage means a 5' nuclease) an upstream oligonucleotide, the cleavage means can be made to cleave a downstream oligonucleotide at an internal site in such a way that the resulting fragments of the downstream oligonucleotide dissociate from the target nucleic acid, thereby making that region of the target nucleic acid available for hybridization to another, uncleaved copy of the downstream oligonucleotide.

As illustrated in Fig. 29, the methods of the present invention employ at least a pair of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for a structure-specific nuclease. More specifically, the cleavage structure comprises i) a target nucleic acid that may be either single-stranded or.double-stranded (when a double-stranded target nucleic acid is employed, it may be rendered single stranded, by heating); ii) a first oligonucleotide, termed the "probe," which defines a first region of the target nucleic acid sequence by being the complement of that region (regions X and Z of the target as shown in Fig. 29); iii) a second oligonucleotide, termed the "invader," the 5' part of which defines a second region of the same target nucleic acid sequence (regions Y and X in Fig. 29), adjacent to and downstream of the first target region (regions X and and the second part of which overlaps into the region defined by the first oligonucleotide (region X depicts the region of .overlap). The resulting structure is diagrammed in Fig. 29.

55 While not limiting the invention or the instant discussion to any particular mechanism of action, the diagram in Fig. 29 represents the effect on the site of cleavage caused by this type of arrangement of a pair of oligonucleotides. The design of such a pair of ligonn.uclotides is descri beblow ii dectaii. in Fig. 29, the 3" ends of the nucleic acids the target and the oligonucleotides) are indicated by the use of the arrowheads on the ends of the lines depicting the strands of the nucleic acids (and where space permits, these ends are also labeled It is readily appreciated that the two oligonucleotides (the invader and the probe) are arranged in a parallel orientation relative to one another, while the target nucleic acid strand is arranged in an anti-parallel orientation relative to the two oligonucleotides.

Further it is clear that the invader oligonucleotide is located upstream of the probe oligonucleotide and that with respect to the target nucleic acid strand, region Z is upstream of region X and region X is upstream of region Y (that is region Y is downstream of region X and region X is downstream of region Regions of complementarity between the opposing strands are indicated by the short vertical lines. While not intended to indicate the precise location of the site(s) of cleavage, the area to which the site of cleavage within the probe oligonucleotide is shifted by the presence of the invader oligonucleotide is indicated by the solid vertical arrowhead. An alternative representation of the target/invader/probe cleavage structure is shown in Fig. 32c. Neither diagram Fig. 29 or Fig. 32c) is intended to represent the actual mechanism of action or physical arrangement of the cleavage structure and further it is not intended that the method of the present invention be limited to any particular mechanism of action.

It can be considered that the binding of these oligonucleotides divides the target nucleic acid into three distinct regions: one region that has complementarity to only the probe (shown as one region that has complementarity only to the invader (shown as and one region that has complementarity to both oligonucleotides (shown as Design of these oligonucleotides the invader and the probe) is accomplished using practices which are standard in the art. For example, sequences that have self complementarity, such that the resulting oligonucleotides would either fold upon themselves, or hybridize to each other at the expense of binding to the target nucleic acid, are generally 30 avoided.

One consideration in choosing a length for these oligonucleotides is the complexity of the sample containing the target nucleic acid. For example, the human genome is approximately 3 x 10' basepairs in length. Any 10 nucleotide sequence will appear with a 56-

.J

frequency of 1:4'1, or 1:1048,576 in a random string of nucleotides, which would be approximately 2,861 times in 3 billion basepairs. Clearly an oligonucleotide of this length would have a poor chance of binding uniquely to a 10 nucleotide region within a target having a sequence the size of the human genome. If the target sequence were within a 3 kb plasmid, however, such an oligonucleotide might have a very reasonable chance of binding uniquely. By this same calculation it can be seen that an oligonucleotide of 16 nucleotides a 16-mer) is the minimum length of a sequence which is mathematically likely to appear once in 3 x 109 basepairs.

A second consideration in choosing oligonucleotide length is the temperature range in which the oligonucleotides will be expected to function. A 16-mer of average base content G-C basepairs) will have a calculated T, (the temperature at which 50% of the sequence is dissociated) of about 41°C, depending on, among other things, the concentration of the oligonucleotide and its target, the salt content of the reaction and the precise order of the nucleotides. As a practical matter, longer oligonucleotides are usually chosen to enhance the specificity of hybridization. Oligonucleotides 20 to 25 nucleotides in length are often used as they are highly likely to be specific if used in reactions conducted at temperatures which are near their Tns (within about 50 of the In addition, with calculated Ts in the range of 50° to 70 0 C, such oligonucleotides 20 to 25-mers) are appropriately used in reactions catalyzed by thermostable enzymes, which often display optimal activity near this 20 temperature range.

The maximum length of the oligonucleotide chosen is also based on the desired specificity. One must avoid choosing sequences that are so long that they are either at a high risk of binding stably to partial complements, or that they cannot easily be dislodged when :desired failure to disassociate from the target once cleavage has occurred).

The first step of design and selection of the oligonucleotides for the invader-directed cleavage is in accordance with these sample general principles. Considered as sequencespecific probes individually, each oligonucleotide may be selected according to the guidelines listed above. That is to say, each oligonucleotide will generally be long enough to be reasonably expected to hybridize only to the intended target sequence within a complex sample, usually in the 20 to 40 nucleotide range. Alternatively, because the invader-directed .cleavage assay depends upon the concerted action of these oligonucleotides, the composite length of the 2 oligonucleotides which span/bind to the X, Y, Z regions may be selected to fall within this range, with each of the individual oligonucleotides being in approximately the 57t 13 to 17 nucleotide range. Such a design might be employed if a non-thermostable cleavage means were employed in the reaction, requiring the reactions to be conducted at a lower temperature than that used when thermostable cleavage means are employed. In some instances, it may be desirable to havc these uligonucieotides bind multiple times within a target nucleic acid which bind to multiple variants or multiple similar sequences within a target). It is not intended that the method of the present invention be limited to any particular size of the probe or invader oligonucleotide.

The second step of designing an oligonucleotide pair for this assay is to choose the degree to which the upstream "invader" oligonucleotide sequence will overlap into the downstream "probe" oligonucleotide sequence, and consequently, the sizes into which the probe will be cleaved. A key feature of this assay is that the probe oligonucleotide can be made to "turn over," that is to say cleaved probe can be made to depart to allow the binding and cleavage of other copies of the probe molecule, without the requirements of thermal denaturation or displacement by polymerization. While in one embodiment of this assay probe turnover may be facilitated by an exonucleolytic digestion by the cleavage agent, it is central to the present invention that the turnover does not reguire this exonucleolytic activity.

1. Choosing The Amount Of Overlap (Length Of The X Region) One way of accomplishing such turnover can be envisioned by considering the diagram in Fig. 29. It can be seen that the Tm of each oligonucleotide will be a function of the full length of that oligonucleotide: the Tm of the invader Tm(Y+X), and the Tm of the probe Tm~x.Y) for the probe. When the probe is cleaved the X region is released, leaving the Z section. If the Tm of Z is less than the reaction temperature, and the reaction .temperature is less than the Tm(x,z), then cleavage of the probe will lead to the departure of Z, thus allowing a new to hybridize. It can be seen from this example that the X region must be sufficiently long that the release of X will drop the Tm of the remaining probe section below the reaction temperature: a G-C rich X section may be much shorter than an A-T rich X section and still accomplish this stability shift.

30 2. Designing Oligonucleotides Which Interact With The Y And Z Regions If the binding of the invader oligonucleotide to the target is more stable than the binding of the probe if it is long, or is rich in G-C basepairs in the Y region), then the copy of X associated with the invader may be favored in the competition for binding to the X -58 region of the target, and the probe may consequently hybridize inefficiently, and the assay may give low signal. Alternatively, if the probe binding is particularly strong in the Z region, the invader will still cause internal cleavage, because this is mediated by the en.zyme, but portion of the probe oligonucleotide bound to the Z region may not dissociate at the reaction temperature, turnover may be poor, and the assay may again give low signal.

It is clearly beneficial for the portions of the oligonucleotide which interact with the Y and Z regions so be similar in stability, they must have similar melting temperatures.

This is not to say that these regions must be the same length. As noted above, in addition to length, the melting temperature will also be affected by the base content and the specific sequence of those bases. The specific stability designed into the invader and probe sequences will depend on the temperature at which one desires to perform the reaction.

This discussion is intended to illustrate that (within the basic guidelines for oligonucleotide specificity discussed above) it is the balance achieved between the stabilities of the probe and invader sequences and their X and Y component sequences, rather than the absolute values of these stabilities, that is the chief consideration in the selection of the probe and invader sequences.

3. Design Of The Reaction Conditions Target nucleic acids that may be analyzed using the methods of the present invention which employ a 5' nuclease as the cleavage means include many types of both RNA and DNA. Such nucleic acids may be obtained using standard molecular biological techniques.

For example. nucleic acids (RNA or DNA) may be isolated from a tissue sample a biopsy specimen), tissue culture cells, samples containing bacteria and/or viruses (including cultures of bacteria and/or viruses), etc. The target nucleic acid may also be transcribed in vitro from a DNA template or may be chemically synthesized or generated in a PCR.

°Furthermore, nucleic acids may be isolated from an organism, either as genomic material or as a plasmid or similar extrachromosomal DNA, or they may be a fragment of such material generated by treatment with a restriction endonuclease or other cleavage agents or it may be synthetic.

Assembly of the target, probe, and invader nucleic acids into the cleavage reaction of the present invention uses principles commonly used in the design of oligonucleotide base enzymatic assays, such as dideoxynucleotide sequencing and polymerase chain reaction (PCR).

As is done in these assays, the oligonucleotides are provided in sufficient excess that the rate 59of hybridization to the target nucleic acid is very rapid. These assays are commonly performed with 50 fmoles to 2 pmoles of each oligonucleotide per lI of reaction mixture. In the Examples described herein, amounts of oligonucleotides ranging from 250 fmoles to pmoies per pi of reaction volume were used. These values were chosen for the purpose of ease in demonstration and are not intended to limit the performance of the present invention to these concentrations. Other lower) oligonucleotide concentrations commonly used in other molecular biological reactions are also contemplated.

It is desirable that an invader oligonucleotide be immediately available to direct the cleavage of each probe oligonucleotide that hybridizes to a target nucleic acid. For this reason, in the Examples described herein, the invader oligonucleotide is provided in excess over the probe oligonucleotide; often this excess is 10-fold. While this is an effective ratio. it is not intended that the practice of the present invention be limited to any particular ratio of invader-to-probe (a ratio of 2- to 100-fold is contemplated).

Buffer conditions must be chosen that will be compatible with both the oligonucleotide/target hybridization and with the activity of the cleavage agent. The optimal buffer conditions for nucleic acid modification enzymes, and particularly DNA modification enzymes, generally included enough mono- and di-valent salts to allow association of nucleic acid strands by base-pairing. If the method of the present invention is performed using an enzymatic cleavage agent other than those specifically described here, the reactions may generally be performed in any such buffer reported to be optimal for the nuclease function of the cleavage agent. In general, to test the utility of any cleavage agent in this method, test reactions are performed wherein the cleavage agent of interest is tested in the MOPS/MnCI 2 /KCI buffer or Mg-containing buffers described herein and in whatever buffer has been reported to be suitable for use with that agent, in a manufacturer's data sheet, a journal article, or in personal communication.

The products of the invader-directed cleavage reaction are fragments generated by structure-specific cleavage of the input oligonucleotides. The resulting cleaved and/or uncleaved oligonucleotides may be analyzed and resolved by a number of methods including electrophoresis (on a variety of supports including acrylamide or agarose gels, paper, etc.), 30 chromatography, fluorescence polarization, mass spectrometry and chip hybridization. The invention is illustrated using electrophoretic separation for the analysis of the products of the cleavage reactions. However, it is noted that the resolution of the cleavage products is not limited to electrophoresis. Electrophoresis is chosen to illustrate the method of the invention because electrophoresis is widely practiced in the art and is easily accessible to the average practitioner.

The probe and invader olignnuclentides may contain a label to aid in their deeciiun following the cleavage reaction. The label may be a radioisotope a nP or "S-labelled nucleotide) placed at either the 5' or 3' end of the oligonucleotide or alternatively, the label may be distributed throughout the oligonucleotide a uniformly labeled oligonucleotide).

The label may be a nonisotopic detectable moiety, such as a fluorophore, which can be detected directly, or a reactive group which permits specific recognition by a secondary agent.

For example, biotinylated oligonucleotides may be detected by probing with a streptavidin molecule which is coupled to an indicator alkaline phosphatase or a fluorophore) or a hapten such as dioxigenin may be detected using a specific antibody coupled to a similar indicator.

4. Optimization Of Reaction Conditions The invader-directed cleavage reaction is useful to detect the presence of specific nucleic acids. In addition to the considerations listed above for the selection and design of the invader and probe oligonucleotides, the conditions under which the reaction is to be performed may be optimized for detection of a specific target sequence.

One objective in optimizing the invader-directed cleavage assay is to allow specific detection of the fewest copies of a target nucleic acid. To achieve this end, it is desirable that the combined elements of the reaction interact with the maximum efficiency, so that the rate of the reaction the number of cleavage events per minute) is maximized. Elements contributing to the overall efficiency of the reaction include the rate of hybridization, the rate of cleavage, and the efficiency of the release of the cleaved probe.

The rate of cleavage will be a function of the cleavage means chosen, and may be made optimal according to the manufacturer's instructions when using commercial preparations of enzymes or as described in the examples herein. The other elements (rate of hybridization, efficiency of release) depend upon the execution of the reaction, and optimization of these elements is discussed below.

Three elements of the cleavage reaction that significantly affect the rate of nucleic acid hybridization are the concentration of the nucleic acids, the temperature at which the cleavage reaction is performed and the concentration of salts and/or other charge-shielding ions in the reaction solution.

-61 The concentrations at which oligonucleotide probes are used in assays of this type are well known in the art, and are discussed above. One example of a common approach to optimizing an oligonucleotide concentration is to choose a starting amount of oligonucleotide .for pilot tests; 0.01 to 2 r i. a uiuciinraiion range used in many oligonucleotide-based assays. When initial cleavage reactions are performed, the following questions may be asked of the data: Is the reaction performed in the absence of the target nucleic acid substantially free of the cleavage product?; Is the site of cleavage specifically shifted in accordance with the design of the invader oligonucleotide?; Is the specific cleavage product easily detected in the presence of the uncleaved probe (or is the amount of uncut material overwhelming the chosen visualization method)? A negative answer to any of these questions would suggest that the probe concentration is too high, and that a set of reactions using serial dilutions of the probe should be performed until the appropriate amount is identified. Once identified for a given target nucleic acid in a give sample type purified genomic DNA, body fluid extract, lysed bacterial extract), it should not need to be re-optimized. The sample type is important because the complexity of the material present may influence the probe optimum.

Conversely, if the chosen initial probe concentration is too low, the reaction may be slow, due to inefficient hybridization. Tests with increasing quantities of the probe will identify the point at which the concentration exceeds the optimum. Since the hybridization will be facilitated by excess of probe, it is desirable, but not required, that the reaction be performed using probe concentrations just below this point.

SThe concentration of invader oligonucleotide can be chosen based on the design considerations discussed above. In a preferred embodiment, the invader oligonucleotide is in excess of the probe oligonucleotide. In a particularly preferred embodiment, the invader is approximately 10-fold more abundant than the probe.

Temperature is also an important factor in the hybridization of oligonucleotides. The range of temperature tested will depend in large part, on the design of the oligonucleotides, as discussed above. In a preferred embodiment, the reactions are performed at temperatures slightly below the T, of the least stable oligonucleotide in the reaction. Melting temperatures 30 for the oligonucleotides and for their component regions Y and Z, Fig. 29), can be estimated through the use of computer software or, for a more rough approximation, by assigning the value of 2 0 C per A-T basepair, and 4 0 C per G-C basepair, and taking the sum across an expanse of nucleic acid. The latter method may be used for oligonucleotides of 62 approximately 10-30 nucleotides in length. Because even computer prediction of the Tm of a nucleic acid is only an approximation, the reaction temperatures chosen for initial tests should bracket the calculated While optimizations are not limited to this, 5°C increments are convenient test intervals in these optimization assays.

When temperatures are tested, the results can be analyzed for specificity (the first two of the questions listed above) in the same way as for the oligonucleotide concentration determinations. Non-specific cleavage cleavage of the probe at many or all positions along its length) would indicate non-specific interactions between the probe and the sample material, and would suggest that a higher temperature should be employed. Conversely, little or no cleavage would suggest that even the intended hybridization is being prevented, and would suggest the use of lower temperatures. By testing several temperatures, it is possible to identify an approximate temperature optimum, at which the rate of specific cleavage of the probe is highest. If the oligonucleotides have been designed as described above, the Tm of the Z-region of the probe oligonucleotide should be below this temperature, so that turnover is assured.

A third determinant of hybridization efficiency is the salt concentration of the reaction.

In large part, the choice of solution conditions will depend on the requirements of the cleavage agent, and for reagents obtained commercially, the manufacturer's instructions are a resource for this information. When developing an assay utilizing any particular cleavage 20 agent, the oligonucleotide and temperature optimizations described above should be performed in the buffer conditions best suited to that cleavage agent.

A "no enzyme" control allows the assessment of the stability of the labeled oligonucleotides under particular reaction conditions, or in the presence of the sample to be tested in assessing the sample for contaminating nucleases). In this manner, the substrate and oligonucleotides are placed in a tube containing all reaction components, except the e enzyme and treated the same as the enzyme-containing reactions. Other controls may also be included. For example, a reaction with all of the components except the target nucleic acid will serve to confirm the dependence of the cleavage on the presence of the target sequence.

:•63 63 Probing For Multiple Alleles The invader-directed cleavage reaction is also useful in the detection and quantification of individual variants or alleles in a mixed sample population. By way of example, such a need ex i e sof t.r r:iaLl Ifir iiuii uiuns in genes associated with cancers.

Biopsy material from a tumor can have a significant complement of normal cells, so it is desirable to detect mutations even when present in fewer than 5% of the copies of the target nucleic acid in a sample. In this case, it is also desirable to measure what fraction of the population carries the mutation. Similar analyses may also be done to examine allelic variation in other gene systems, and it is not intended that the method of the present invention by limited to the analysis of tumors.

As demonstrated below, reactions can be performed under conditions that prevent the cleavage of probes bearing even a single-nucleotide difference mismatch within the region of the target nucleic acid termed in Fig. 29, but that permit cleavage of a similar probe that is completely complementary to the target in this region. Thus, the assay may be used to quantitate individual variants or alleles within a mixed sample.

The use of multiple, differently labeled probes in such an assay is also contemplated.

To assess the representation of different variants or alleles in a sample, one would provide a mixture of probes such that each allele or variant to be detected would have a specific probe perfectly matched to the Z region of the target sequence) with a unique label no two variant probes with the same label would be used in a single reaction). These probes would be characterized in advance to ensure that under a single set of reaction conditions, 4 •they could be made to give the same rate of signal accumulation when mixed with their respective target nucleic acids. Assembly of a cleavage reaction comprising the mixed probe set, a corresponding invader oligonucleotide, the target nucleic acid sample, and the appropriate cleavage agent, along with performance of the cleavage reaction under conditions such that only the matched probes would cleave, would allow independent quantification of each of the species present, and would therefore indicate their relative representation in the target sample.

-64- IV. A Comparison Of Invasive Cleavage And Primer-Directed Cleavage As discussed herein, the terms "invasive" or "invader-directed" cleavage specifically denote the use of a first, upstream oligonucleotide, as defined below, to cause soecific cleavage at a site within a second, downstream sequence. To effect such a direction of cleavage to a region within a duplex, it is required that the first and second oligonucleotides overlap in sequence. That is to say, a portion of the upstream oligonucleotide, termed the "invader", has significant homology to a portion of the downstream "probe" oligonucleotide, so that these regions would tend to basepair with the same complementary region of the target nucleic acid to be detected. While not limiting the present invention to any particular mechanism, the overlapping regions would be expected to alternate in their occupation of the shared hybridization site. When the probe oligonucleotide fully anneals to the target nucleic acid, and thus forces the 3' region of the invader to remain unpaired, the structure so formed is not a substrate for the 5' nucleases of the present invention. By contrast, when the inverse is true, the structure so formed is substrate for these enzymes, allowing cleavage and release of the portion of the probe oligonucleotide that is displaced by the invader oligonucleotide.

The shifting of the cleavage site to a region the probe oligonucleotide that would otherwise be basepaired to the target sequence is one hallmark of the invasive cleavage assay the invader-directed cleavage assay) of the present invention.

It is beneficial at this point to contrast the invasive cleavage as described above with 20 two other forms of probe cleavage that may lead to internal cleavage of a probe oligonucleotide, but which do not comprise invasive cleavage. In the first case, a hybridized probe may be subject to duplex-dependent 5' to 3' exonuclease "nibbling," such that the oligonucleotide is shortened from the 5' end until it cannot remain bound to the target (see, Examples 6-8 and Figs. 26-28). The site at which such nibbling stops can appear to be discrete, and, depending on the difference between the melting temperature of the full-length probe and the temperature of the reaction, this stopping point may be 1 or several nucleotides into the probe oligonucleotide sequence. Such "nibbling" is often indicated by the presence of a "ladder" of longer products ascending size up to that of the full length of the probe, but this is not always the case. While any one of the products of such a nibbling reaction may be made to match in size and cleavage site the products of an invasive cleavage reaction, the creation of these nibbling products would be highly dependent on the temperature of the reaction and the nature of the cleavage agent, but would be independent of the action of an upstream oligonucleotide, and thus could not be construed to involve invasive cleavage.

A second cleavage structure that may be considered is one in which a probe oligonucleotide has several regions of complementarity with the target nucleic acid, interspersed with one or more regions or nucleotides of noncomplementarity. These noncomplementarv regions may be thought of as "bubbles" within the nucieic acid duplex.

As temperature is elevated, the regions of complementarity can be expected to "melt" in the order of their stability, lowest to highest. When a region of lower stability is near the end of a segment of duplex, and the next region of complementarity along the strand has a higher melting temperature, a temperature can be found that will cause the terminal region of duplex to melt first, opening the first bubble, and thereby creating a preferred substrate structure of the cleavage by the 5' nucleases of the present invention (Fig. 40a). The site of such cleavage would be expected to be on the 5' arm, within 2 nucleotides of the junction between the single and double-stranded regions (Lyamichev et al.. supra. and U.S. Patent No.

5,422,253) An additional oligonucleotide could be introduced to basepair along the target nucleic acid would have a similar effect of opening this bubble for subsequent cleavage of the unpaired 5' arm (Fig. 40b and Fig. Note in this case, the 3' terminal nucleotides of the upstream oligonucleotide anneals along the target nucleic acid sequence in such a manner that the 3' end is located within the "bubble" region. Depending on the precise location of the 3' end of this oligonucleotide, the cleavage site may be along the newly unpaired 5' arm, or at the site expected for the thermally opened bubble structure as described above. In the former *case the cleavage is not within a duplexed region, and is thus not invasive cleavage, while in S* the latter the oligonucleotide is merely an aide in inducing cleavage at a site that might otherwise be exposed through the use of temperature alone in the absence of the additional oligonucleotide), and is thus not considered to be invasive cleavage.

In summary, any arrangement of oligonucleotides used for the cleavage-based detection of a target sequence can be analyzed to determine if the arrangement is an invasive cleavage structure as contemplated herein. An invasive cleavage structure supports cleavage of the probe in a region that, in the absence of an upstream oligonucleotide, would be expected to be basepaired to the target nucleic acid.

30 Ex. 26 below provides further guidance for the design and execution of a experiments which allow the determination of whether a given arrangement of a pair of upstream and downstream the probe) oligonucleotides when annealed along a target nucleic acid would form an invasive cleavage structure.

-66- V. Fractionation Of Specific Nucleic Acids By Selective Charge Reversal Some nucleic acid-based detection assays involve the elongation and/or shortening of oligonucleotide probes. For example, as described herein, the primer-directed, primerindependent, and invader-directed cleavage assays, as well as the "nibbling" assay all involve the cleavage shortening) of oligonucleotides as a means for detecting the presence of a target nucleic sequence. Examples of other detection assays which involve the shortening of an oligonucleotide probe include the "TaqMan" or nick-translation PCR assay described in U.S. Patent No. 5,210,015 to Gelfand et al. (the disclosure of which is herein incorporated by reference), the assays described in U.S. Patent Nos. 4,775,619 and 5,118,605 to Urdea (the disclosures of which are herein incorporated by reference), the catalytic hybridization amplification assay described in U.S. Patent No. 5,403,711 to Walder and Walder (the disclosure of which is herein incorporated by reference), and the cycling probe assay described in U.S. Patents Nos. 4,876,187 and 5,011,769 to Duck et al. (the disclosures of which are herein incorporated by reference). Examples of detection assays which involve the elongation of an oligonucleotide probe (or primer) include the polymerase chain reaction (PCR) described in U.S. Patent Nos. 4,683,195 and 4,683.202 to Mullis and Mullis et al. (the disclosures of which are herein incorporated by reference) and the ligase chain reaction (LCR) described in U.S. Patent Nos. 5.427,930 and 5,494,810 to Birkenmeyer et al. and Barany et al. (the disclosures of which are herein incorporated by reference). The above examples are 20 intended to be illustrative of nucleic acid-based detection assays that involve the elongation and/or shortening of oligonucleotide probes and do not provide an exhaustive list.

Typically, nucleic acid-based detection assays that involve the elongation and/or shortening of oligonucleotide probes require post-reaction analysis to detect the products of the reaction. It is common that, the specific reaction product(s) must be separated from the other reaction components, including the input or unreacted oligonucleotide probe. One .detection technique involves the electrophoretic separation of the reacted and unreacted oligonucleotide probe. When the assay involves the cleavage or shortening of the probe, the unreacted product will be longer than the reacted or cleaved product. When the assay involves the elongation of the probe (or primer), the reaction products will be greater in 30 length than the input. Gel-based electrophoresis of a sample containing nucleic acid molecules of different lengths separates these fragments primarily on the basis of size. This is due to the fact that in solutions having a neutral or alkaline pH, nucleic acids having widely different sizes molecular weights) possess very similar charge-to-mass ratios and do not 67 separate (Andrews, Electrophoresis, 2nd Edition, Oxford University Press [1986], pp. 153- 154). The gel matrix acts as a molecular sieve and allows nucleic acids to be separated on the basis of size and shape linear, relaxed circular or covalently closed supercoiled Unmodified nucleic acids have a net negative charge due to the presence of negatively charged phosphate groups contained within the sugar-phosphate backbone of the nucleic acid.

Typically, the sample is applied to gel near the negative pole and the nucleic acidc fragments migrate into the gel toward the positive pole with the smallest fragments moving fastest through the gel.

The present invention provides a novel means for fractionating nucleic acid fragments on the basis of charge. This novel separation technique is related to the observation that positively charged adducts can affect the electrophoretic behavior of small oligonucleotides because the charge of the adduct is significant relative to charge of the whole complex. In addition, to the use of positively charged adducts Cy3 and Cy5 amidite fluorescent dyes, the positively charged heterodimeric DNA-binding dyes shown in Fig. 66, etc.), the oligonucleotide may contain amino acids (particularly useful amino acids are the charged amino acids: lysine, arginine, asparate, glutamate), modified bases, such as amino-modified bases, and/or a phosphonate backbone (at all or a subset of the positions). In addition as discussed further below, a neutral dye or detection moiety biotin, streptavidin, etc.) may be employed in place of a positively charged adduct in conjunction with the use of amino-modified bases and/or a complete or partial phosphonate backbone.

This observed effect is of particular utility in assays based on the cleavage of DNA molecules. Using the assays described herein as an example, when an oligonucleotide is *shortened through the action of a Cleavase® enzyme or other cleavage agent, the positive charge can be made to not only significantly reduce the net negative charge, but to actually override it, effectively "flipping" the net charge of the labeled entity. This reversal of charge allows the products of target-specific cleavage to be partitioned from uncleaved probe by extremely simple means. For example, the products of cleavage can be made to migrate towards a negative electrode placed at any point in a reaction vessel, for focused detection 30 without gel-based electrophoresis; Ex. 24 provides examples of devices suitable for focused detection without gel-based electrophoresis. When a slab gel is used, sample wells can be S* positioned in the center of the gel, so that the cleaved and uncleaved probes can be observed to migrate in opposite directions. Alternatively, a traditional vertical gel can be used, but -68 with the electrodes reversed relative to usual DNA gels the positive electrode at the top and the negative electrode at the bottom) so that the cleaved molecules enter the gel, while the uncleaved disperse into the upper reservoir of electrophoresis buffer.

An important benefit of this type of readout is the absolute nature of the partition of products from substrates, the separation is virtually 100%. This means that an abundance of uncleaved probe can be supplied to drive the hybridization step of the probe-based assay, yet the unconsumed unreacted) probe can, in essence, be subtracted from the result to reduce background by virtue of the fact that the unreacted probe will not migrate to the same pole as the specific reaction product.

Through the use of multiple positively charged adducts, synthetic molecules can be constructed with sufficient modification that the normally negatively charged strand is made nearly neutral. When so constructed, the presence or absence of a single phosphate group can mean the difference between a net negative or a net positive charge. This observation has particular utility when one objective is to discriminate between enzymatically generated fragments of DNA, which lack a 3' phosphate, and the products of thermal degradation, which retain a 3' phosphate (and thus two additional negative charges). Examples 23 and 24 demonstrate the ability to separate positively charged reaction products from a net negatively charged substrate oligonucleotide. As discussed in these examples, oligonucleotides may be transformed from net negative to net positively charged compounds. In Ex. 24, the positively 20 charged dye, Cy3 was incorporated at the 5' end of a 22-mer (SEQ ID NO:61) which also contained two amino-substituted residues at the 5' end of the oligonucleotide; this oligonucleotide probe carries a net negative charge. After cleavage, which occurred 2 nucleotides into the probe, the following labeled oligonucleotide was released: 5'-Cy3- AminoT-AminoT-3'(as well as the remaining 20 nucleotides of SEQ ID NO:61). This short fragment bears a net positive charge while the remainder of the cleaved oligonucleotide and the unreacted or input oligonucleotide bear net negative charges.

The present invention contemplates embodiments wherein the specific reaction product produced by any cleavage of any oligonucleotide can be designed to carry a net positive •charge while the unreacted probe is charge neutral or carries a net negative charge. The 30 present invention also contemplates embodiments where the released product may be designed to carry a net negative charge while the input nucleic acid carries a net positive charge.

SDepending on the length of the released product to be detected, positively charged dyes may be incorporated at the one end of the probe and modified bases may be placed along the 69 oligonucleotide such that upon cleavage, the released fragment containing the positively charged dye carries a net positive charge. Amino-modified bases may be used to balance the charge of the released fragment in cases where the presence of the positively charged adduct dyc) alone is not sufficient to impart a net positive charge on the released fragment. In addition, the phosphate backbone may be replaced with a phosphonate backbone at a level sufficient to impart a net positive charge (this is particularly useful when the sequence of the oligonucleotide is not amenable to the use of amino-substituted bases); Figs. 56 and 57 show the structure of short oligonucleotides containing a phosphonate group on the second T residue). An oligonucleotide containing a fully phosphonate-substituted backbone would be charge neutral (absent the presence of modified charged residues bearing a charge or the presence of a charged adduct) due to the absence of the negatively charged phosphate groups.

Phosphonate-containing nucleotides methylphosphonate-containing nucleotides are readily available and can be incorporated at any position of an oligonucleotide during synthesis using techniques which are well known in the art.

In essence, the invention contemplates the use of charge-based separation to permit the separation of specific reaction products from the input oligonucleotides in nucleic acid-based detection assays. The foundation of this novel separation technique is the design and use of oligonucleotide probes (typically termed "primers" in the case of PCR) which are "charge balanced" so that upon either cleavage or elongation of the probe it becomes "charge unbalanced," and the specific reaction products may be separated from the input reactants on the basis of the net charge.

In the context of assays which involve the elongation of an oligonucleotide probe o a primer), such as is the case in PCR, the input primers are designed to carry a net positive charge. Elongation of the short oligonucleotide primer during polymerization will generate PCR products which now carry a net negative charge. The specific reaction products may then easily be separated and concentrated away from the input primers using the charge-based separation technique described herein (the electrodes will be reversed relative to the description in Ex. 24, as the product to be separated and concentrated after a PCR will carry a negative charge).

VI. InvaderTM-Directed Cleavage Using Miniprobes And Mid-Range Probes As discussed in section III above, the InvaderTM-directed cleavage assay may be performed using invader and probe oligonucleotides which have a length of about 13-25 uc'i tids (typically 20-25 nucieotides). it is also contemplated that the oligonucleotides that span the X. Y and Z regions (see Fig. 29), the invader and probe oligonucleotides, may themselves be composed of shorter oligonucleotide sequences that align along a target strand but that are not covalently linked. This is to say that there is a nick in the sugaf-phosphate backbone of the composite oligonucleotide, but that there is no disruption in the progression of base-paired nucleotides in the resulting duplex. When short strands of nucleic acid align contiguously along a longer strand the hybridization of each is stabilized by the hybridization of the neighboring fragments because the basepairs can stack along the helix as though the backbone was in fact uninterrupted. This cooperativity of binding can give each segment a stability of interaction in excess of what would be expected for the segment hybridizing to the longer nucleic acid alone. One application of this observation has been to assemble primers for DNA sequencing, typically about 18 nucleotides long, from sets of three hexamer oligonucleotides that are designed to hybridize in this way (Kotler et al., Proc. Natl. Acad.

Sci. USA 90:4241 [1993]). The resulting doubly-nicked primer can be extended enzymatically in reactions performed at temperatures that might be expected to disrupt the hybridization of hexamers, but not of 18-mers.

The use of composite or split oligonucleotides is applied with success in the SInvaderTM-directed cleavage assay. The probe oligonucleotide may be split into two oligonucleotides which anneal in a contiguous and adjacent manner along a target o* oligonucleotide as diagrammed in Fig. 68. In this figure, the downstream oligonucleotide O. (analogous to the probe of Fig. 29) is assembled from two smaller pieces: a short segment of 6-10 nts (termed the "miniprobe"), that is to be cleaved in the course of the detection reaction, and an oligonucleotide that hybridizes immediately downstream of the miniprobe (termed the 9 "stacker"), which serves to stabilize the hybridization of the probe. To form the cleavage structure, an upstream oligonucleotide (the "InvaderTM" oligonucleotide) is provided to direct the cleavage activity to the desired region of the miniprobe. Assembly of the probe from 30 non-linked pieces of nucleic acid the miniprobe and the stacker) allows regions of sequences to be changed without requiring the re-synthesis of the entire proven sequence, thus improving the cost and flexibility of the detection system. In addition, the use of unlinked composite oligonucleotides makes the system more stringent in its requirement of perfectly 71 i matched hybridization to achieve signal generation, allowing this to be used as a sensitive means of detecting mutations or changes in the target nucleic acid sequences.

As illustrated in Fig. 68, in one embodiment, the methods of the present invention employ at fleast three cigonuclcotides that intieaci with a target nucleic acid to form a cleavage structure for a structure-specific nuclease. More specifically, the cleavage structure comprises i) a target nucleic acid that may be either single-stranded or double-stranded (when a double-stranded target nucleic acid is employed, it may be rendered single-stranded, by heating); ii) a first oligonucleotide, termed the "stacker," which defines a first region of the target nucleic acid sequence by being the complement of that region (region W of the target as shown in Fig. 67); iii) a second oligonucleotide, termed the "miniprobe," which defines a second region of the target nucleic acid sequence by being the complement of that region (regions X and Z of the target as shown in Fig. 67); iv) a third oligonucleotide, termed the "invader," the 5' part of which defines a third region of the same target nucleic acid sequence (regions Y and X in Fig. 67), adjacent to and downstream of the second target region (regions X and and the second or 3' part of which overlaps into the region defined by the second oligonucleotide (region X depicts the region of overlap). The resulting structure is diagrammed in Fig. 68.

While not limiting the invention or the instant discussion to any particular mechanism of action, the diagram in Fig. 68 represents the effect on the site of cleavage caused by this 20 type of arrangement of three oligonucleotides. The design of these three oligonucleotides is

OS

Sdescribed below in detail. In Fig. 68, the 3' ends of the nucleic acids the target and the oligonucleotides) are indicated by the use of the arrowheads on the ends of the lines depicting the strands of the nucleic acids (and where space permits, these ends are also labeled It is readily appreciated that the three oligonucleotides (the invader, the miniprobe and the stacker) are arranged in a parallel orientation relative to one another, while the target nucleic acid strand is arranged in an anti-parallel orientation relative to the three oligonucleotides.

Further it is clear that the invader oligonucleotide is located upstream of the miniprobe oligonucleotide and that the miniprobe oligonucleotide is located upstream of the stacker oligonucleotide and that with respect to the target nucleic acid strand, region W is upstream of 30 region Z, region Z is upstream of upstream of region X and region X is upstream of region Y (that is region Y is downstream of region X, region X is downstream of region Z and region Z is downstream of region Regions of complementarity between the opposing strands are indicated by the short vertical lines. While not intended to indicate the precise location of the 72 site(s) of cleavage, the area to which the site of cleavage within the miniprobe oligonucleotide is shifted by the presence of the invader oligonucleotide is indicated by the solid vertical arrowhead. Fig. 68 is not intended to represent the actual mechanism of action or physical arrangement of the c!cavagc suctur did further it is not intended that the method of the present invention be limited to any particular mechanism of action.

It can be considered that the binding of these oligonucleotides divides the target nucleic acid into four distinct regions: one region that has complementarity to only the stacker (shown as one region that has complementarity to only the miniprobe (shown as one region that has complementarity only to the InvaderTM oligonucleotide (shown as and one region that has complementarity to both the Invader T M and miniprobe oligonucleotides (shown as In addition to the benefits cited above, the use of a composite design for the oligonucleotides which form the cleavage structure allows more latitude in the design of the reaction conditions for performing the InvaderTM-directed cleavage assay. When a longer probe 16-25 nt), as described in section III above, is used for detection in reactions that are performed at temperatures below the Tm of that probe, the cleavage of the probe may play a significant role in destabilizing the duplex of which it is a part, thus allowing turnover and reuse of the recognition site on the target nucleic acid. In contrast, with miniprobes, reaction temperatures that are at or above the Tm of the probe mean that the probe molecules are 20 hybridizing and releasing from the target quite rapidly even without cleavage of the probe.

o When an upstream InvaderTM oligonucleotide and a cleavage means are provided the miniprobe will be specifically cleaved, but the cleavage will not be necessary to the turnover of the miniprobe. If a long probe 16-25 nt) were to be used in this way the temperatures required to achieve this state would be quite high, around 65 to 70 0 C for a mer of average base composition. Requiring the use of such elevated temperatures limits the choice of cleavage agents to those that are very thermostable, and may contribute to *background in the reactions, depending of the means of detection, through thermal degradation of the probe oligonucleotides. Thus, the shorter probes are preferable for use in S. this way.

The miniprobe of the present invention may vary in size depending on the desired application. In one embodiment, the probe may be relatively short compared to a standard probe 16-25 nt), in the range of 6 to 10 nucleotides. When such a short probe is used reaction conditions can be chosen that prevent hybridization of the miniprobe in the absence -73 of the stacker oligonucleotide. In this way a short probe can be made to assume the statistical specificity and selectivity of a longer sequence. In the event of a perturbation in the cooperative binding of the miniprobe and stacker nucleic acids, as might be caused by a mismatch ithin he short sequence (i.e regin which is the region of the miniprobe which does not overlap with the invader) or at the junction between the contiguous duplexes, this cooperativity can be lost, dramatically reducing the stability of the shorter oligonucleotide the miniprobe), and thus reducing the level of cleaved product in the assay of the present invention.

It is also contemplated that probes of intermediate size may be used. Such probes, in the 11 to 15 nucleotide range, may blend some of the features associated with the longer probes as originally described, these features including the ability to hybridize and be cleaved absent the help of a stacker oligonucleotide. At temperatures below the expected T, of such probes, the mechanisms of turnover may be as discussed above for probes in the 20 nt range, and be dependent on the removal of the sequence in the region for destabilization and cycling.

The mid-range probes may also be used at elevated temperatures, at or above their expected Tm, to allow melting rather than cleavage to promote probe turnover. In contrast to the longer probes described above, however, the temperatures required to allow the use of such a thermally driven turnover are much lower (about 40 to 60 0 thus preserving both the 20 cleavage means and the nucleic acids in the reaction from thermal degradation. In this way.

the mid-range probes may perform in some instances like the miniprobes described above. In a further similarity to the miniprobes, the accumulation of cleavage signal from a mid-range probe may be helped under some reaction conditions by the presence of a stacker.

To summarize, a standard long probe usually does not benefit from the presence of a stacker oligonucleotide downstream (the exception being cases where such an oligonucleotide may also disrupt structures in the target nucleic acid that interfere with the probe binding), and it is usually used in conditions requiring several nucleotides to be removed to allow the oligonucleotide to release from the target efficiently.

SThe miniprobe is very short and performs optimally in the presence of a downstream stacker oligonucleotide. The miniprobes are well suited to reactions conditions that use the temperature of the reaction to drive rapid exchange of the probes on the target regardless of whether any bases have been cleaved. In reactions with sufficient amount of the cleavage means, the probes that do bind will be rapidly cleaved before they melt off.

74- The mid-range or midiprobe combines features of these probes and can be used in reactions like those designed long probes, with longer regions of overlap regions) to drive probe turnover at lower temperature. In a preferred embodiment, the midrange probes are used at temperatures sufficiently high that the probes are hybridizing to the target and releasing rapidly regardless of cleavage. This is known to be the behavior of oligonucleotides at or near their melting temperature. This mode of turnover is more similar to that used with miniprobe/stacker combinations than with long probes. The mid-range probe may have enhanced performance in the presence of a stacker under some circumstances. For example, with a probe in the lower end of the mid-range, 11 nt, or one with exceptional A/T content, in a reaction performed well in excess of the Tm of the probe >10°C above) the presence of a stacker would be likely to enhance the performance of the probe, while at a more moderate temperature the probe may be indifferent to a stacker.

The distinctions between the mini-. midi- mid-range) and long probes are not contemplated to be inflexible and based only on length. The performance of any given probe may vary with its specific sequence, the choice of solution conditions, the choice of temperature and the selected cleavage means.

is shown in Ex. 18 that the assemblage of oligonucleotides that comprises the cleavage structure of the present invention is sensitive to mismatches between the probe and the target. The site of the mismatch used in Ex. 18 provides one example and is not intended to be a limitation in location of a mismatch affecting cleavage. It is also contemplated that a mismatch between the InvaderM oligonucleotide and the target may be used to distinguish related target sequences. In the 3-oligonucleotide system, comprising an InvaderT

M

a probe eeeee S°and a stacker oligonucleotide, it is contemplated that mismatches may be located within any of the regions of duplex formed between these oligonucleotides and the target sequence. In a preferred embodiment, a mismatch to be detected is located in the probe. In a particularly preferred embodiment, the mismatch is in the probe, at the basepair immediately upstream of the site that is cleaved when the probe is not mismatched to the target.

.In another preferred embodiment, a mismatch to be detected is located within the So region defined by the hybridization of a miniprobe. In a particularly preferred embodiment, the mismatch is in the miniprobe, at the basepair immediately upstream of the site that is cleaved when the miniprobe is not mismatched to the target.

75 It is also contemplated that different sequences may be detected in a single reaction.

Probes specific for the different sequences may be differently labeled. For example, the probes may have different dyes or other detectable moieties, different lengths, or they may have differences in net charges of the prodimct nfter claage. Nx..n "rr tV l.a'uCid in one of these ways, the contribution of each specific target sequence to final product can be tallied.

This has application in detecting the quantities of different versions of a gene within a mixture. Different genes in a mixture to be detected and quantified may be wild-type and mutant genes, as may be found in a tumor sample a biopsy). In this embodiment, one might design the probes to precisely the same site, but one to match the wild-type sequence and one to match the mutant. Quantitative detection of the products of cleavage from a reaction performed for a set amount of time will reveal the ratio of the two genes in the mixture. Such analysis may also be performed on unrelated genes in a mixture. This type of analysis is not intended to be limited to two genes. Many variants within a mixture may be similarly measured.

Alternatively, different sites on a single gene may be monitored and quantified to verify the measurement of that gene. In this embodiment, the signal from each probe would ,be expected to be the same.

It is also contemplated that multiple probes may be used that are not differently labeled, such that the aggregate signal is measured. This may be desirable when using many 20 probes designed to detect a single gene to boost the signal from that gene. This configuration S. may also be used for detecting unrelated sequences within a mix. For example, in blood banking it is desirable to know if any one of a host of infectious agents is present in a sample of blood. Because the blood is discarded regardless of which agent is present, different signals on the probes would not be required in such an application of the present invention, and may actually be undesirable for reasons of confidentiality.

*Just as described for the two-oligonucleotide system, above, the specificity of the detection reaction will be influenced by the aggregate length of the target nucleic acid sequences involved in the hybridization of the complete set of the detection oligonucleotides.

For example, there may be applications in which it is desirable to detect a single region within a complex genome. In such a case the set of oligonucleotides may be chosen to require accurate recognition by hybridization of a longer segment of a target nucleic acid, often in the range of 20 to 40 nucleotides. In other instances it may be desirable to have the set of oligonucleotides interact with multiple sites within a target sample. In these cases one -76 approach would be to use a set of oligonucleotides that recognize a smaller, and thus statistically more common, segment of target nucleic acid sequence.

In one preferred embodiment, the invader and stacker oligonucleotides may be designed to be maximally stable, so that they will remain bound to the target sequence for extended periods during the reaction. This may be accomplished through any one of a number of measures well known to those skilled in the art, such as adding extra hybridizing sequences to the length of the oligonucleotide (up to about 50 nts in total length),-or by using residues with reduced negative charge, such as phosphorothioates or peptide-nucleic acid residues, so that the complementary strands do not repel each other to degree that natural strands do. Such modifications may also serve to make these flanking oligonucleotides resistant to contaminating nucleases, thus further ensuring their continued presence on the target strand during the course of the reaction. In addition, the InvaderTM and stacker oligonucleotides may be covalently attached to the target through the use of psoralen cross-linking).

The use of the reaction temperatures at or near the Tm of the probe oligonucleotide, rather than the used of cleavage, to drive the turnover of the probe oligonucleotide in these detection reactions means that the amount of the probe oligonucleotide cleaved off may be substantially reduced without adversely affecting the turnover rate. It has been determined that the relationship between the 3' end of the upstream oligonucleotide and the desired site 20 of cleavage on the probe must be carefully designed. It is known that the preferred site of cleavage for the types of structure specific endonucleases employed herein is one basepair into a duplex (Lyamichev et al., supra). It was previously believed that the presence of an upstream oligonucleotide or primer allowed the cleavage site to be shifted away from this preferred site, into the single stranded region of the 5' arm (Lyamichev et al., supra and U.S.

Patent No. 5,422,253, herein incorporated by reference). In contrast to this previously proposed mechanism, and while not limiting the present invention to any particular mechanism, it is believed that the nucleotide immediately or upstream of the cleavage site on the probe (including miniprobe and mid-range probes) must be able to basepair with the target for efficient cleavage to occur. In the case of the present invention, this would be the nucleotide in the probe sequence immediately upstream of the intended cleavage site. In addition, as described herein, it has been observed that in order to direct cleavage to that same site in the probe, the upstream oligonucleotide must have its 3' base nt) immediately upstream of the intended cleavage site of the probe. This places the 3' terminal nucleotide of 77 the upstream oligonucleotide and the base of the probe oligonucleotide 5' of the cleavage site in competition for pairing with the corresponding nucleotide of the target strand.

To examine the outcome of this competition which base is paired during a successful cleavage event), substitutions were made in the probe and invader oligonucleotides such that either the probe or the InvaderTM oligonucleotide were mismatched with the target sequence at this position. The effects of both arrangements on the rates of cleavage were examined. When the InvaderTM oligonucleotide is unpaired at the 3' end, the rateof cleavage was not reduced. If this base was removed, however, the cleavage site was shifted upstream of the intended site. In contrast, if the probe oligonucleotide was not base-paired to the target just upstream of the site to which the InvaderTM oligonucleotide was directing cleavage, the rate of cleavage was dramatically reduced, suggesting that when a competition exists, the probe oligonucleotide was the molecule to be base-paired in this position.

It appears that the 3' end of the upstream invader oligonucleotide is unpaired during cleavage, and yet is required for accurate positioning of the cleavage. To examine which part(s) of the 3' terminal nucleotide are required for the positioning of cleavage, InvaderTM oligonucleotides were designed that terminated on this end with nucleotides that were altered in a variety of ways. Sugars examined included 2' deoxyribose with a 3' phosphate group, a dideoxyribose, 3' deoxyribose. 2' O-methyl ribose, arabinose and arabinose with a 3' phosphate. Abasic ribose, with and without 3' phosphate were tested. Synthetic "universal" bases such at 3-nitropyrrole and 5-3nitroindole on ribose sugars were tested. Finally, a baselike aromatic ring structure, acridine, linked to the 3' end the previous nucleotide without a sugar group was tested. The results obtained support the conclusion that the aromatic ring of •the base (at the 3' end of the invader oligonucleotide) is the required moiety for accomplishing the direction of cleavage to the desired site within the downstream probe.

VII. Signal Enhancement By Tailing Of Reaction Products In The InvaderTM-Directed Cleavage Assay It has been determined that when oligonucleotide probes are used in cleavage detection assays at elevated temperature, some fraction of the truncated probes will have been shortened by nonspecific thermal degradation, and that such breakage products can make the analysis of the target-specific cleavage data more difficult. Background cleavage such as this can, when not resolved from specific cleavage products, reduce the accuracy of quantitation of target nucleic acids based on the amount of accumulated product in a set timeframe. One means of 78 distinguishing the specific from the nonspecific products is disclosed above, and is based on partitioning the products of these reactions by differences in the net charges carried by the different molecular species in the reaction. As was noted in that discussion, the thermal breakage products usually retain 3' phosphates after breakage, while the enzyme-cleaved products do not. The two negative charges on the phosphate facilitate charge-based partition of the products.

The absence of a 3' phosphate on the desired subset of the probe fragments may be used to advantage in enzymatic assays as well. Nucleic acid polymerases, both non-templated terminal deoxynucleotidyl transferase, polyA polymerase) and template-dependent Pol I-type DNA polymerases), require an available 3' hydroxyl by which to attach further nucleotides. This enzymatic selection of 3' end structure may be used as an effective means of partitioning specific from non-specific products.

In addition to the benefits of the partitioning described above, the addition of nucleotides to the end of the specific product of an invader-specific cleavage offers an opportunity to either add label to the products, to add capturable tails to facilitate solid-support based readout systems, or to do both of these things at the same time. Some possible embodiments of this concept are illustrated in Fig. 67.

In Fig. 67, an InvaderTM cleavage structure comprising an InvaderTM oligonucleotide containing a blocked or non-extendible 3' end a 3' dideoxynucleotide) and a probe 20 oligonucleotide containing a blocked or non-extendible 3' end (the open circle at the 3' end of the oligonucleotides represents a non-extendible nucleotide) and a target nucleic acid is shown; the probe oligonucleotide may contain a 5' end label such as a biotin or a fluorescein (indicated by the stars) label (cleavage structures which employ a 5' biotin-labeled probe or a fluorescein-labeled probe are shown below the large diagram of the cleavage structure to 25 the left and the right, respectively). Following, cleavage of the probe (the site of cleavage is indicated by the large arrowhead), the cleaved biotin-labeled probe is extended using a template-independent polymerase TdT) and fluoresceinated nucleotide triphosphates.

The fluorescein tailed cleaved probe molecule is then captured by binding via its 5' biotin label to streptavidin and the fluorescence is then measured. Alternatively, following, cleavage of a 5'-fluoresceinated probe, the cleaved probe is extended using a template-independent polymerase TdT) and dATP. The polyadenylated (A-tailed) cleaved probe molecule is then captured by binding via the polyA tail to oligonucleotide dT attached to a solid support.

79 The examples described in Fig. 66 are based on the use of TdT to tail the specific products of InvaderT'M-directed cleavage. The description of the use of this particular enzyme is presented by way of example and is not intended as a limitation (indeed, when probe oligonucleotides comprising RNA are employed, cleaved RNA probes may be extended using polyA polymerase). It is contemplated that an assay of this type could be configured to use a template-dependent polymerase, as described above. While this would require the presence of a suitable copy template distinct from the target nucleic acid, on which the truncated oligonucleotide could prime synthesis, it can be envisaged that a probe which before cleavage would be unextendible, due to either mismatch or modification of the 3' end, could be activated as a primer when cleaved by an invader directed cleavage. A template directed tailing reaction also has the advantage of allowing greater selection and control of the nucleotides incorporated.

The use of nontemplated tailing does not require the presence of any additional nucleic acids in the detection reaction, avoiding one step of assay development and troubleshooting.

In addition, the use of non templated synthesis eliminated the step of hybridization, potentially speeding up the assay. Furthermore, the TdT enzyme is fast, able to add at least >700 .nucleotides to substrate oligonucleotides in a 15 minute reaction.

iAs mentioned above, the tails added can be used in a number of ways. It can be used as a straight-forward way of adding labeled moieties to the cleavage product to increase signal from each cleavage event. Such a reaction is depicted in the left side of Fig. 66. The labeled moieties may be anything that can, when attached to a nucleotide, be added by the tailing enzyme, such as dye molecules, haptens such as digoxigenin, or other binding groups such as biotin.

In a preferred embodiment the assay includes a means of specifically capturing or partitioning the tailed invader-directed cleavage products in the mixture. It can be seen that target nucleic acids in the mixture may be tailed during the reaction. If a label is added, it is desirable to partition the tailed invader-directed cleavage products from these other labeled molecules to avoid background in the results. This is easily done if only the cleavage product is capable of being captured. For example, consider a cleavage assay of the present invention in which the probe used has a biotin on the 5' end and is blocked from extension on the 3' end, and in which a dye is added during tailing. Consider further that the products are to be captured onto a support via the biotin moiety, and the captured dye measured to assess the presence of the target nucleic acid. When the label is added by tailing, only the specifically

"A

cleaved probes will be labeled. The residual uncut probes can still bind in the final capture step, but they will not contribute to the signal. In the same reaction, nicks and cuts in the target nucleic acid may be tailed by the enzyme, and thus become dye labeled. In the final capture these labeled targets will not bind to the support and thus, though labeled, they will not contribute to the signal. If the final specific product is considered to consist of two portions, the probe-derived portion and the tail portion, can be seen from this discussion that it is particularly preferred that when the probe-derived portion is used for specific capture, whether by hybridization, biotin/streptavidin, or other method, that the label be associated with the tail portion. Conversely, if a label is attached to the probe-derived portion, then the tail portion may be made suitable for capture, as depicted on the right side of Fig. 66. Tails may be captured in a number of ways, including hybridization, biotin incorporation with streptavidin capture, or by virtue if the fact that the longer molecules bind more predictably and efficiently to a number of nucleic acid minding matrices, such as nitrocellulose, nylon, or glass, in membrane, paper, resin, or other form. While not required for this assay, this separation of functions allows effective exclusion from signal of both unreacted probe and tailed target nucleic acid.

In addition to the supports described above, the tailed products may be captured onto S• any support that contains a suitable capture moiety. For example, biotinylated products are i generally captured with avidin-treated surfaces. These avidin surfaces may be in microtitre plate wells, on beads, on dipsticks, to name just a few of the possibilities. Such surfaces can S"also be modified to contain specific oligonucleotides, allowing capture of product by hybridization. Capture surfaces as described here are generally known to those skilled in the art and include nitrocellulose dipsticks GeneComb, BioRad, Hercules, CA).

-25 VIII. Improved Enzymes For Use In InvaderTM-Directed Cleavage Reactions A cleavage structure is defined herein as a structure which is formed by the interaction of a probe oligonucleotide and a target nucleic acid to form a duplex, the resulting structure being cleavable by a cleavage means, including but not limited to an enzyme. The cleavage structure is further defined as a substrate for specific cleavage by the cleavage means in contrast to a nucleic acid molecule which is a substrate for nonspecific cleavage by agents such as phosphodiesterases. Examples of some possible cleavage structures are shown in Fig.

16. In considering improvements to enzymatic cleavage means, one may consider the action of said enzymes on any of these structures, and on any other structures that fall within the -81-

I

definition of a cleavage structure. The cleavage sites indicated on the structures in Fig. 16 are presented by way of example. Specific cleavage at any site within such a structure is contemplated.

Improvements in an enzyme may be an increased or decreased rate of cleavage of one or more types of structures. Improvements may also result in more or fewer sites of cleavage on one or more of said cleavage structures. In developing a library of new structure-specific nucleases for use in nucleic acid cleavage assays, improvements may have many different embodiments, each related to the specific substrate structure used in a particular assay.

As an example, one embodiment of the InvaderTM-directed cleavage assay of the present invention may be considered. In the InvaderTM directed cleavage assay, the accumulation of cleaved material is influenced by several features of the enzyme behavior.

Not surprisingly, the turnover rate, or the number of structures that can be cleaved by a single enzyme molecule in a set amount of time, is very important in determining the amount of material processed during the course of an assay reaction. If an enzyme takes a long time to recognize a substrate if it is presented with a less-than-optimal structure), or if it takes a long time to execute cleavage, the rate of product accumulation is lower than if these steps proceeded quickly. If these steps are quick, yet the enzyme "holds on" to the cleaved structure, and does not immediately proceed to another uncut structure, the rate will be ~negatively affected.

Enzyme turnover is not the only way in which enzyme behavior can negatively affect the rate of accumulation of product. When the means used to visualize or measure product is specific for a precisely defined product, products that deviate from that definition may escape eeeo° detection, and thus the rate of product accumulation may appear to be lower than it is. For example, if one had a sensitive detector for trinucleotides that could not see di- or tetranucleotides, or any sized oligonucleotide other that 3 residues, in the InvaderTM-directed cleavage assay of the present invention any errant cleavage would reduce the detectable signal proportionally. It can be seen from the cleavage data presented here that, while there is usually one site within a probe that is favored for cleavage, there are often products that arise from cleavage one or more nucleotides away from the primary cleavage site. These are products that are target dependent, and are thus not non-specific background. Nevertheless, if a subsequent visualization system can detect only the primary product, these represent a loss of signal. One example of such a selective visualization system is the charge reversal readout presented herein, in which the balance of positive and negative charges determines the 82 behavior of the products. In such a system the presence of an extra nucleotide or the absence of an expected nucleotide can excluded a legitimate cleavage product from ultimate detection by leaving that product with the wrong balance of charge. It can be easily seen that any assay uhat can sensitively distinguish the nucleotide content of an oligonucleotide, such as standard stringent hybridization, suffers in sensitivity when some fraction of the legitimate product is not eligible for successful detection by that assay.

These discussions suggest two highly desirable traits in any enzyme to be used in the method of the present invention. First, the more rapidly the enzyme executes an entire cleavage reaction, including recognition, cleavage and release, the more signal it may potentially created in the invader-directed cleavage assay. Second, the more successful an enzyme is at focusing on a single cleavage site within a structure, the more of the cleavage product can be successfully detected in a selective read-out.

The rationale cited above for making improvements in enzymes to be used in the InvaderTM-directed cleavage assay are meant to serve as an example of one direction in which improvements might be sought, but not as a limit on either the nature or the applications of improved enzyme activities. As another direction of activity change that would be appropriately considered improvement, the DNAP-associated 5' nucleases may be used as an example. In creating some of the polymerase-deficient 5' nucleases described herein it was •"found that the those that were created by deletion of substantial portions of the polymerase 20 domain, as depicted in Fig. 4, assumed activities that were weak or absent in the parent proteins. These activities included the ability to cleave the non-forked structure shown in Fig.

16D, a greatly enhanced ability to exonucleolytically remove nucleotides from the 5' ends of •duplexed strands, and a nascent ability to cleave circular molecules without benefit of a free 5' end. These features have contributed to the development of detection assays such as the one depicted in Fig. IA.

83 IX. Improved Enzymes For Use in The CFLP® Method As defined herein, a folded cleavage structure is a single-stranded nucleic acid molecule which contains one or more regions containing secondary structure, said region(s) be...g cea.'bc by a cleavage ineans, including but not limited to an enzyme. The folded cleavage structure is further defined as a substrate for specific cleavage by said cleavage means in contrast to a nucleic acid molecule which is a substrate for nonspecific cleavage by agents such as phosphodiesterases. Examples of some possible cleavage structures are shown in Fig. 16.

In considering improvements to enzymatic cleavage means, the action of structurespecific enzymes on any of these folded structures, and on any other structures that fall within the definition of a cleavage structure may be taken into consideration. The cleavage sites indicated on the structures in Fig. 16 are presented by way of example. The present invention contemplates specific cleavage at any site within such a structure. It is also contemplated that improvements of an enzyme may encompass an increased or decreased rate of cleavage of one or more types of structures folded or otherwise. Improvements may also result in more or fewer sites of cleavage on one or more of cleavage structures. In developing a library of new V or additional structure-specific nucleases for use in nucleic acid cleavage assays, improvements may have many different embodiments, each related to the specific substrate structure used in a particular assay.

20 As an example, one embodiment of the CFLPV assay of the present invention may be considered. In the CFLP® assay, the distribution of cleavage products is influenced by several features of the enzyme's behavior. In some instances it has been observed that DNAs •eoeo a DNA substrate) may have regions of sequence that are resistant to cleavage by a particular structure-specific nuclease, such as the Cleavase® BN nuclease or other nucleases 25 of the present invention. Although an understanding of the mechanisms is not necessary in eeoo* order to use the present invention, these regions may be unstructured lack secondary structure), or the structures that form are not well recognized by these nucleases. In the latter case, it is contemplated that the use of a nuclease that does cleave in such a region will allow So additional existing structural information to be visualized. The data presented in Examples 38 and 40 demonstrate this particular advantage provided through use of an alternative nuclease in CFLP® analysis, one with either a different substrate recognition specificity, so that a different set of structures are represented in the cleavage pattern.

84 In addition, there may be cases in which no single enzyme nuclease) provides sufficient cleavage across an entire nucleic acid fragment of interest. An optimal cleavage pattern is considered to be one in which the bands are evenly spaced, the bands are as even in intensity as possible, and the fragment sizes run from full-length (to assure representation of the larger fragments) down to the size of the shortest cleavable fragment (about 15 to 20 nt).

More detailed patterns ones that contain many bands about 15) rather than a few bands about are also more likely to show differences in response to sequence changes a single point mutation between two forms of a substrate, such as a mutant and a wildtype form). When the spacing and intensity distribution of a CFLP® pattern, or the cleavage frequency number of bands) of a fragment of interest is not optimal using a single enzyme, it is contemplated that in some embodiments of the present invention, mixtures of enzymes provides the desired enhancement of the cleavage pattern. The enhancing effect of mixing nucleases which have slightly different substrate specificities is demonstrated in the experiment shown in Fig. 100.

In addition to the 5' nucleases derived from DNA polymerases, the present invention also contemplates the use of structure-specific nucleases that are not derived from DNA polymerases. For example, a class of eukaryotic and archaebacterial endonucleases have been identified which have a similar substrate specificity to 5' nucleases of Pol I-type DNA polymerases. These are the FEN-I (Flap EndoNuclease), RAD2, and XPG (Xeroderma 20 Pigmentosa-complementation group G) proteins. These proteins are involved in DNA repair.

and have been shown to favor the cleavage of structures that resemble a 5' arm that has been displaced by an extending primer during polymerization, similar to the model depicted in Fig.

S"16B. Similar DNA repair enzymes have been isolated from single cell and higher eukaryotes and from archaea, and there are related DNA repair proteins in eubacteria. Similar nucleases have also be associated with bacteriophage such as T5 and T7.

Surprisingly, and contrary to reports in the literature, it is demonstrated herein that the FEN-1 proteins can also effectively cleavage folded structures similar to that depicted in Fig.

16A. This type of structure is the structure formed when nucleic acids assume secondary structures. Literature defining the FEN class of nucleases states that no activity is observed on either single strands of DNA, or on structures lacking a primer upstream of the cleavage site (such as that in Fig. 16A). (See Harrington and Lieber, supra).

Recently, the 3-dimensional structures of DNAPTaq and T5 phage (Fig. 69) were determined by X-ray diffraction (Kim et al., Nature 376:612 [1995]and Ceska 85 et al., Nature 382:90 [1995]). The two enzymes have very similar 3-dimensional structures despite limited amino acid sequence similarity. The most striking feature of the structure is the existence of a triangular hole formed by the active site of the proiein and two alpha heiices (Fig. 69). This same region of DNAPTaq is disordered in the crystal structure, indicating that this region is flexible, and thus is not shown in the published 3-dimensional structure. However, the 5' nuclease domain of DNAPTaq is likely to have the same structure, based its overall 3-dimensional similarity to T5 5'-exonuclease, ana that the amino acids in the disordered region of the DNAPTaq protein are those associated with alpha helix formation. The existence of such a hole or groove in the 5' nuclease domain of DNAPTaq was predicted based on its substrate specificity (Lyamichev et al., supra).

It has been suggested that the 5' arm of a cleavage structure must thread through the helical arch described above to position said structure correctly for cleavage (Ceska et al., supra). One of the modifications of 5' nucleases described herein opened up the helical arch portion of the protein to allow improved cleavage of structures that cut poorly or not at all structures on circular DNA targets that would preclude such threading of a 5' arm).

The gene construct that was chosen as a model to test this approach was the one called Cleavase® BN, which was derived from DNAPTaq but does not contain the polymerase domain (See, Ex. It comprises the entire 5' nuclease domain of DNAP Taq, and thus should be very close in structure to the T5 5' exonuclease. This 5' nuclease was chosen to demonstrate the principle of such a physical modification on proteins of this type. The arch-opening modification of the present invention is not intended to be limited to the nuclease domains of DNA polymerases, and is contemplated for use on any structure-specific nuclease which includes such an aperture as a limitation on cleavage activity. The present invention contemplates the insertion of a thrombin cleavage site into the helical arch of 25 DNAPs derived from the genus Thermus as well as 5' nucleases derived from DNAPs derived from the genus Thermus. The specific example shown herein using the Cleavase® BN/thrombin nuclease merely illustrates the concept of opening the helical arch located within a nuclease domain. As the amino acid sequence of DNAPs derived from the genus Thermus are highly conserved, the teachings of the present invention enable the insertion of a thrombin site into the helical arch present in these DNAPs and 5' nucleases derived from these DNAPs.

The opening of the helical arch was accomplished by insertion of a protease site in the arch. This allowed post-translational digestion of the expressed protein with the appropriate protease to open the arch at its apex. Proteases of this type recognize short stretches of 86specific amino acid sequence. Such proteases include thrombin and factor Xa. Cleavage of a protein with such a protease depends on both the presence of that site in the amino acid sequence of the protein and the accessibility of that site on the folded intact protein. Even with a crystal structure it can be difficult to predict the susceptibility of any particular region of a protein to protease cleavage. Absent a crystal structure it must be determined empirically.

In selecting a protease for a site-specific cleavage of a protein that has been modified to contain a protease cleavage site, a first step is to test the unmodified protein for cleavage at alternative sites. For example, DNAPTaq and Cleavase® BN nuclease were both incubated under protease cleavage conditions with factor Xa and thrombin proteases. Both nuclease proteins were cut with factor Xa within the 5' nuclease domain, but neither nuclease was digested with large amounts of thrombin. Thus, thrombin was chosen for initial tests on opening the arch of the Cleavase® BN enzyme.

In the protease/Cleavase® modifications described herein the factor Xa protease cleaved strongly in an unacceptable position in the unmodified nuclease protein, in a region likely to compromise the activity of the end product. Other unmodified nucleases contemplated herein may not be sensitive to the factor Xa, but may be sensitive to thrombin or other such proteases. Alternatively, they may be sensitive to these or other such proteases at sites that are immaterial to the function of the nuclease sought to be modified. In 20 approaching any protein for modification by addition of a protease cleavage site, the unmodified protein should be tested with the proteases under consideration to determine which proteases give acceptable levels of cleavage in other regions.

S*Working with the cloned segment of DNAPTaq from which the Cleavase® BN protein is expressed, nucleotides encoding a thrombin cleavage site were introduced in-frame near the sequence encoding amino acid 90 of the nuclease gene. This position was determined to be at or near the apex of the helical arch by reference to both the 3-dimensional structure of DNAPTaq, and the structure of T5 5' exonuclease.

The encoded amino acid sequence, LVPRGS, was inserted into the apex of the helical arch by site-directed mutagenesis of the nuclease gene. The proline in the thrombin cleavage site was positioned to replace a proline normally in this position in Cleavase® BN because proline is an alpha helix-breaking amino acid, and may be important for the 3-dimensional structure of this arch. This construct was expressed, purified and then digested with thrombin. The digested enzyme was tested for its ability to cleave a target nucleic acid, 87 bacteriophage M13 genomic DNA, that does not provide free 5' ends to facilitate cleavage by the threading model.

While the helical arch in this nuclease was opened by protease cleavage, it is contem,-.platd that a br of other tcuciliques couid be used to achieve the same end. For example, the nucleotide sequence could be rearranged such that, upon expression, the resulting protein would be configured so that the top of the helical arch (amino acid 90) would be at the amino terminus of the protein, the natural carboxyl and amino termini of the-protein sequence would be joined, and the new carboxyl terminus would lie at natural amino acid 89.

This approach has the benefit that no foreign sequences are introduced and the enzyme is a single amino acid chain, and thus may be more stable that the cleaved 5' nuclease. In the crystal structure of DNAPTaq, the amino and carboxyl termini of the 5'-exonuclease domain lie in close proximity to each other, which suggests that the ends may be directly joined without the use of a flexible linker peptide sequence as is sometimes necessary. Such a rearrangement of the gene, with subsequent cloning and expression could be accomplished by standard PCR recombination and cloning techniques known to those skilled in the art.

The present invention also contemplates the use of nucleases isolated from a organisms that grow under a variety of conditions. The genes for the FEN-1/XPG class of enzymes are found in organisms ranging from bacteriophage to humans to the extreme thermophiles of -o Kingdom Archaea. For assays in which high temperature is to be used, it is contemplated that 20 enzymes isolated from extreme thermophiles may exhibit the thermostability required of such an assay. For assays in which it might be desirable to have peak enzyme activity at moderate temperature or in which it might be desirable to destroy the enzyme with elevated temperature, those enzymes from organisms that favor moderate temperatures for growth may be of particular value.

An alignment of a collection of FEN-1 proteins sequenced by others is shown in Figs.

It can be seen from this alignment that there are some regions of conservation in this class of proteins, suggesting that they are related in function, and possibly in structure.

Regions of similarity at the amino acid sequence level can be used to design primers for in vitro amplification (PCR) by a process of back translating the amino acid sequence to the possible nucleic acid sequences, then choosing primers with the fewest possible variations within the sequences. These can be used in low stringency PCR to search for related DNA sequences. This approach permits the amplification of DNA encoding a FEN-1 nuclease without advance knowledge of the actual DNA sequence.

88 It can also be seen from this alignment that there are regions in the sequences that are not completely conserved. The degree of difference observed suggests that the proteins may have subtle or distinct differences is substrate specificity. In other words, they may have different levels of cleavage activity on the cleavage structures of the present invention. When a particular structure is cleaved at a higher rate than the others, this is referred to a preferred substrate, while a structure that is cleaved slowly is considered a less preferred substrate. The designation of preferred or less preferred substrates in this context is not intended to be a limitation of the present invention. It is contemplated that some embodiments the present invention will make use of the interactions of an enzyme with a less preferred substrate.

Candidate enzymes are tested for suitability in the cleavage assays of the present invention using the assays described below.

As noted above, the variations in structure-specific nucleases from organism to organism, or between different natural structure specific nucleases within a single organism can lead to the observation of different activities in both the test systems and the final assay applications of these nucleases. It has been noted that it may be desirable in some cases to use two or more of these nucleases in combination, within a single assay to make use of the ~combines abilities of the proteins. It is also noted above that many of these nucleases have similar morphological features when crystal structures are compared, even when the primary nucleic acid and amino acid sequences are quite divergent.

It is contemplated that the functions of the nucleases of the present invention may be •modified and improved by combining different regions of the proteins within a single nuclease molecule. One method in which this may be accomplished is by the construction e e•• S•and expression of genes containing the coding regions for the desired portions of each protein.

While it is not intended to limit the structure and/or composition of such nucleases, it is 25 contemplated that chimerical nucleases may be composed of portions derived from two distinct natural enzymes. It is further contemplated that portions of more than two natural enzymes may by combined in a single chimeric nuclease.

o The conservation of structure of the 5' nucleases allows the primary sequences to be aligned sufficiently that regions that are likely to have the same function within the protein can be identified. While these common regions may perform similar functions, this is not to say that the functions are performed with identical mechanisms or with the same performance characteristics turnover rate, substrate Kmin, ion requirements). For example, the segment of sequence likely to correspond to the "loop region" described herein can be located on each 89 protein sequence, by both location within the amino acid sequence, and by conservation within the amino acid sequence. As used in this instance, conservation in the amino acid sequence comprises not only precise amino acid identity, but also resulting character of the sequence, including but no limi- ted iu itydrophobicity, hydrophilicity, positive or negative charge, a-helix or p-sheet forming nature or steric features.

The portions of sequence used to make the chimeric nucleases of the present invention may be chosen such that the final construct has a single representative of each putative domain. In a preferred embodiment the sequence domains are arranged so as to mimic the domain alignments of the natural enzymes.

The improvements to be gained by the creation of chimeric nucleases are not limited to any particular feature of such a nuclease. As described above, improvements in an enzyme may include an increased or decreased rate of cleavage of one or more types of structures.

Improvements may also result in more or fewer sites of cleavage on one or more of said cleavage structures. Improvements may also result in changes in the stability of the enzyme with or without any changes in the cleavage functions cited above. In developing a library of new structure-specific nucleases for use in nucleic acid cleavage assays, improvements may have many different embodiments, each related to the specific substrate structure used in a particular assay.

pturs 1. Structure-Specific Nuclease Assay Testing candidate nucleases for structure-specific activities in these assays is done in '*much the same way as described for testing modified DNA polymerases in Ex. 2, but with the use of a different library of model structures. In addition to assessing the enzyme °performance in primer-independent and primer-directed cleavage, a set of synthetic hairpins are used to examine the length of duplex downstream of the cleavage site preferred by the enzyme.

The FEN-1 and XPG 5' nucleases used in the present invention must be tested for S*activity in the assays in which they are intended to be used, including but not limited to the InvaderTM-directed cleavage detection assay of the present invention and the CFLP® method of characterizing nucleic acids (the CFLP® method is described in co-pending Application Serial Nos. 08/337,164, 08/402,601, 08/484,956 and 08/520,946; the disclosures of these applications are incorporated herein by reference). The InvaderTM assay uses a mode of cleavage that has been termed "primer directed" of "primer dependent" to reflect the influence of the an oligonucleotide hybridized to the target nucleic acid upstream of the cleavage site.

90 In contrast, the CFLP® reaction is based on the cleavage of folded structure, or hairpins, within the target nucleic acid, in the absence of any hybridized oligonucleotide. The tests described herein are not intended to be limited to the analysis of nucleases with any particular site of cleavage or mode of recognition of substrate structures. It is contemplated that enzymes may be described as 3' nucleases, utilizing the 3' end as a reference point to recognize structures, or may have a yet a different mode of recognition. Further, the use of the term 5' nucleases is not intended to limit consideration to enzymes that cleave the cleavage structures at any particular site. It refers to a general class of enzymes that require some reference or access to a 5' end to effect cleavage of a structure.

A set of model cleavage structures have been created to allow the cleavage ability of unknown enzymes on such structures to be assessed. Each of the model structures is constructed of one or more synthetic oligonucleotides made by standard DNA synthesis chemistry. Examples of such synthetic model substrate structures are shown in Figs. 30 and 71. These are intended only to represent the general folded configuration desirable is such test structures. While a sequence that would assume such a structure is indicated in the figures, there are numerous other sequence arrangements of nucleotides that would be expected to fold in such ways. The essential features to be designed into a set of ooligonucleotides to perform the tests described herein are the presence or absence of a I sufficiently long 3' arm to allow hybridization of an additional nucleic acid to test cleavage in 20 a "primer-directed" mode, and the length of the duplex region. In the set depicted in Fig. 71, the duplex lengths of the S-33 and the 11-8-0 structures are 12 and 8 basepairs, respectively.

This difference in length in the test molecules facilitates detection of discrimination by the candidate nuclease between longer and shorter duplexes. Additions to this series expanding the range of duplex molecules presented to the enzymes, both shorter and longer, may be used. The use of a stabilizing DNA tetraloop (Antao et al., Nucl. Acids Res. 19:5901 [1991]) or triloop (Hiraro et al., Nuc. Acids Res. 22:576 [1994]) at the closed end of the duplex helps ensure formation of the expected structure by the oligonucleotide.

The effects of specific modifications on the activities of the nucleases of the present invention may be assessed by making such modifications in a test structure. For example, positively charged moieties such as Cy3 dye and amine groups may be added to the 5' end of a probe to be cleaved, in order to assess the performance of these cleavage agents in the charge reversal method described above and in Ex. 23.

-91 The model substrate for testing primer directed cleavage, the "S-60 hairpin" (SEQ ID is described in Ex. 11 In the absence of a primer this hairpin is usually cleaved to release 5' arm fragments of 18 and 19 nucleotides length. An oligonucleotide, termed P-14 (5'-CGAGAGACCACGCT-3')(SFQ ID N:122), that vextends to th base of the dup. x wh...

hybridized to the 3' arm of the S-60 hairpin gives cleavage products of the same size, but at a higher rate of cleavage.

To test invasive cleavage a different primer is used, termed P-15 CGAGAGACCACGCTG-3')(SEQ ID NO:41). In a successful invasive cleavage the presence of this primer shifts the site of cleavage of S-60 into the duplex region, usually releasing products of 21 and 22 nucleotides length.

The S-60 hairpin may also be used to test the effects of modifications of the cleavage structure on either primer-directed or invasive cleavage. Such modifications include, but are not limited to, use of mismatches or base analogs in the hairpin duplex at one, a few or all positions, similar disruptions or modifications in the duplex between the primer and the 3' arm of the S-60, chemical or other modifications to one or both ends of the primer sequence, or attachment of moieties to, or other modifications of the 5' arm of the structure. In all of the analyses using the S-60 or a similar hairpin described herein, activity with and without a primer may be compared using the same hairpin structure.

0 The assembly of these test reactions, including appropriate amounts of hairpin, primer 20 and candidate nuclease are described in Ex. 2. As cited therein, the presence of cleavage products is indicated by the presence of molecules which migrate at a lower molecular weight than does the uncleaved test structure. When the reversal of charge of a label is used the products will carry a different net charge than the uncleaved material. Any of these cleavage products indicate that the candidate nuclease has the desired structure-specific nuclease activity. By "desired structure-specific nuclease activity" it is meant only that the candidate nuclease cleaves one or more test molecules. It is not necessary that the candidate nuclease cleave at any particular rate or site of cleavage to be considered successful cleavage.

-92-

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: Afu (Archaeoglobus filgidus); Mth (Methanobacterium thermoautotrophicum); Mja (Methanococcus jannaschii); Pfu (Pyrococcus furiosus); Pwo (Pyrococcus woesei); Taq (Thermus aquaticus); Taq DNAP, DNAPTaq, and Taq Pol I aquaticus DNA polymerase DNAPStf (the Stoffel fragment of DNAPTaq); DNAPEcl coli DNA polymerase I); Tth (Thermus thermophilus); Ex. (Example); Fig. (Figure);°C (degrees Centigrade); g (gravitational field); vol (volume); w/v (weight to volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB (cetyltrimethylammonium bromide); HPLC (high pressure liquid chromatography); DNA (deoxyribonucleic acid); p (plasmid); pl (microliters); ml (milliliters); pg (micrograms); pmoles (picomoles); mg (milligrams); M (molar); mM (milliMolar); pM (microMolar); nm (nanometers); kdal (kilodaltons); OD (optical density); 15 EDTA (ethylene diamine tetra-acetic acid); FITC (fluorescein isothiocyanate); SDS (sodium dodecyl sulfate); NaPO 4 (sodium phosphate); Tris (tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride); TBE (Tris-Borate-EDTA, Tris buffer titrated with boric acid rather than HCI and containing EDTA) PBS (phosphate buffered saline); PPBS (phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamide gel electrophoresis); Tween (polyoxyethylene-sorbitan); ATCC (American Type Culture Collection, Rockville, MD); DSMZ (Deutsche Sammlung von Mikroorganismen und Zellculturen, Braunschweig, Germany); Sigma (Sigma Chemical Company, St. Louis, MO); Dynal (Dynal Oslo, Norway); Gull (Gull Laboratories, Salt Lake City, UT); Epicentre (Epicentre Technologies, Madison, WI); MJ Research (MJ Research, Watertown,MA); 25 National Biosciences (Plymouth, MN); New England Biolabs (Beverly, MA); Novagen (Novagen, Inc., Madison, WI); Perkin Elmer (Norwalk, CT); Promega Corp. (Madison, WI); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Clonetech (Clonetech, Palo Alto, CA) Pharmacia (Pharmacia, Piscataway, NJ); Milton Roy (Milton Roy, Rochester, NY); Amersham (Amersham International, Chicago, IL); and USB Biochemical, Cleveland, OH).

93 EXAMPLE 1 Characteristics Of Native Thermostable DNA Polymerases A. 5' Nuclease Activity Of DNAPTaq D-ring the pov.ymcrasc chain reaction (PCR) (Saiki el al., Science 239:487 [1988]; Mullis and Faloona, Methods in Enzymology 155:335 [1987]), DNAPTaq is able to amplify many, but not all, DNA sequences. One sequence that cannot be amplified using DNAPTaq is shown in Fig. 6 (Hairpin structure is SEQ ID NO:15, PRIMERS are SEQ ID NOS:16-17.) This DNA sequence has the distinguishing characteristic of being able to fold on itself to form a hairpin with two single-stranded arms, which correspond to the primers used in PCR.

To test whether this failure to amplify is due to the 5' nuclease activity of the enzyme, we compared the abilities of DNAPTaq and DNAPStf to amplify this DNA sequence during cycles of PCR. Synthetic oligonucleotides were obtained from The Biotechnology Center at the University of Wisconsin-Madison. The DNAPTaq and DNAPStf were from Perkin Elmer Amplitaq TM DNA polymerase and the Stoffel fragment of AmplitaqTM DNA polymerase). The substrate DNA comprised the hairpin structure shown in Fig. 6 cloned in a double-stranded form into pUC19. The primers used in the amplification are listed as SEQ ID NOS:16-17. Primer SEQ ID NO:17 is shown annealed to the 3' arm of the hairpin structure in Fig. 6. Primer SEQ ID NO:16 is shown as the first 20 nucleotides in bold on the 5' arm of the hairpin in Fig. 6.

Polymerase chain reactions comprised 1 ng of supercoiled plasmid target DNA, pmoles of each primer, 40 pM each dNTP, and 2.5 units of DNAPTaq or DNAPStf, in a solution of 10 mM Tris*Cl pH 8.3. The DNAPTaq reactions included 50 mM KCI and .mM MgCl 2 The temperature profile was 95 0 C for 30 sec., 55 0 C for 1 min. and 72 0 C for 1 min., through 30 cycles. Ten percent of each reaction was analyzed by gel electrophoresis 25 through 6% polyacrylamide (cross-linked 29:1) in a buffer of 45 mM Tris*Borate, pH 8.3, 1.4 mM EDTA.

The results are shown in Fig. 7. The expected product was made by DNAPStf (indicated simply as but not by DNAPTaq (indicated as We conclude that the nuclease activity of DNAPTaq is responsible for the lack of amplification of this DNA sequence.

To test whether the 5' unpaired nucleotides in the substrate region of this structured DNA are removed by DNAPTaq, the fate of the end-labeled 5' arm during four cycles of PCR was compared using the same two polymerases (Fig.. The hairpin templates, such as -94the one described in Fig. 6, were made using DNAPStf and a 3 P-5'-end-labeled primer. The of the DNA was released as a few large fragments by DNAPTaq but not by DNAPStf.

The sizes of these fragments (based on their mobilities) show that they contain most or all of tihe unpaired 5' arm of the DNA. Thus, cleavage occurs at or near the base of the bifurcated duplex. These released fragments terminate with 3' OH groups, as evidenced by direct sequence analysis, and the abilities of the fragments to be extended by terminal deoxynucleotidyl transferase.

Figs. 9-11 show the results of experiments designed to characterize the cleavage reaction catalyzed by DNAPTaq. Unless otherwise specified, the cleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeled hairpin DNA (with the unlabeled complementary strand also present), 1 pmole primer (complementary to the 3' arm) and units of DNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10.l of 10 mM Tris- Cl, pH 8.5, 50 mM KCI and 1.5 mM MgCI 2 As indicated, some reactions had different concentrations of KCI, and the precise times and temperatures used in each experiment are 15 indicated in the individual figures. The reactions that included a primer used the one shown in Fig. 6 (SEQ ID NO:17). In some instances, the primer was extended to the junction site by providing polymerase and selected nucleotides.

Reactions were initiated at the final reaction temperature by the addition of either the MgCl, or enzyme. Reactions were stopped at their incubation temperatures by the addition of 8 pl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. The Tm calculations listed were made using the Oligo

T

M primer analysis software from National Biosciences, Inc.

These were determined using 0.25 lM as the DNA concentration, at either 15 or 65 mM total salt (the 1.5 mM MgCI 2 in all reactions was given the value of 15 mM salt for these calculations).

25 Fig. 9 is an autoradiogram containing the results of a set of experiments and conditions on the cleavage site. Fig. 9A is a determination of reaction components that enable cleavage.

SIncubation of 5'-end-labeled hairpin DNA was for 30 minutes at 55 0 C, with the indicated components. The products were resolved by denaturing polyacrylamide gel electrophoresis and the lengths of the products, in nucleotides, are indicated. Fig. 9B describes the effect of temperature on the site of cleavage in the absence of added primer. Reactions were incubated in the absence of KCI for 10 minutes at the indicated temperatures. The lengths of the products, in nucleotides, are indicated.

95 Surprisingly, cleavage by DNAPTaq requires neither a primer nor dNTPs (see Fig.

9A). Thus, the 5' nuclease activity can be uncoupled from polymerization. Nuclease activity requires magnesium ions, though manganese ions can be substituted, albeit with potential changes in specificity and activity. Neither zinc nor calcium ions support the cleavage reaction. The reaction occurs over a broad temperature range, from 25 0 C to 85 0 C, with the rate of cleavage increasing at higher temperatures.

Still referring to Fig. 9, the primer is not elongated in the absence of added dNTPs.

However, the primer influences both the site and the rate of cleavage of the hairpin. The change in the site of cleavage (Fig. 9A) apparently results from disruption of a short duplex formed between the arms of the DNA substrate. In the absence of primer, the sequences indicated by underlining in Fig. 6 could pair, forming an extended duplex. Cleavage at the end of the extended duplex would release the 11 nucleotide fragment seen on the Fig. 9A lanes with no added primer. Addition of excess primer (Fig. 9A, lanes 3 and 4) or incubation at an elevated temperature (Fig. 9B) disrupts the short extension of the duplex and results in a longer 5' arm and. hence, longer cleavage products.

:The location of the 3' end of the primer can influence the precise site of cleavage.

Electrophoretic analysis revealed that in the absence of primer (Fig. 9B), cleavage occurs at "•the end of the substrate duplex (either the extended or shortened form, depending on the temperature) between the first and second base pairs. When the primer extends up to the base of the duplex, cleavage also occurs one nucleotide into the duplex. However, when a gap of :o-o four or six nucleotides exists between the 3' end of the primer and the substrate duplex, the cleavage site is shifted four to six nucleotides in the 5' direction.

Fig. 10 describes the kinetics of cleavage in the presence (Fig. 10A) or absence (Fig.

of a primer oligonucleotide. The reactions were run at 55C with either 50 mM KCI (Fig. 10A) or 20 mM KCI (Fig. lOB). The reaction products were resolved by denaturing polyacrylamide gel electrophoresis and the lengths of the products, in nucleotides, are indicated. indicating a marker, is a 5' end-labeled 19-nt oligonucleotide. Under these salt conditions, Figs. IOA and 10B indicate that the reaction appears to be about twenty times faster in the presence of primer than in the absence of primer. This effect on the efficiency may be attributable to proper alignment and stabilization of the enzyme on the substrate.

The relative influence of primer on cleavage rates becomes much greater when both reactions are run in 50 mM KCI. In the presence of primer, the rate of cleavage increases with KCI concentration, up to about 50 mM. However, inhibition of this reaction in the -96presence of primer is apparent at 100 mM and is complete at 150 mM KCI. In contrast, in the absence of primer the rate is enhanced by concentration of KCI up to 20 mM, but it is reduced at concentrations above 30 mM. At 50 mM KCL. the reaction is almost completely inhibited. The inhibition of cleavage by KCI in the absence of primer is affec:cd by temperature, being more pronounced at lower temperatures.

Recognition of the 5' end of the arm to be cut appears to be an important feature of substrate recognition. Substrates that lack a free 5' end, such as circular M13 DNA, cannot be cleaved under any conditions tested. Even with substrates having defined 5' arms, the rate of cleavage by DNAPTaq is influenced by the length of the arm. In the presence of primer and 50 mM KCI, cleavage of a 5' extension that is 27 nucleotides long is essentially complete within 2 minutes at 55 0 C. In contrast, cleavages of molecules with 5' arms of 84 and 188 nucleotides are only about 90% and 40% complete after 20 minutes. Incubation at higher temperatures reduces the inhibitory effects of long extensions indicating that secondary structure in the 5' arm or a heat-labile structure in the enzyme may inhibit the reaction. A mixing experiment, run under conditions of substrate excess, shows that the molecules with long arms do not preferentially tie up the available enzyme in non-productive complexes.

These results may indicate that the 5' nuclease domain gains access to the cleavage site at the end of the bifurcated duplex by moving down the 5' arm from one end to the other. Longer 5' arms would be expected to have more adventitious secondary structures (particularly when 20 KCI concentrations are high), which would be likely to impede this movement.

Cleavage does not appear to be inhibited by long 3' arms of either the substrate strand target molecule or pilot nucleic acid, at least up to 2 kilobases. At the other extreme. 3' arms of the pilot nucleic acid as short as one nucleotide can support cleavage in a primerindependent reaction, albeit inefficiently. Fully paired oligonucleotides do not elicit cleavage of DNA templates during primer extension.

The ability of DNAPTaq to cleave molecules even when the complementary strand contains only one unpaired 3' nucleotide may be useful in optimizing allele-specific PCR.

PCR primers that have unpaired 3' ends could act as pilot oligonucleotides to direct selective cleavage of unwanted templates during preincubation of potential template-primer complexes with DNAPTaq in the absence of nucleoside triphosphates.

97 B. 5' Nuclease Activities Of Other DNAPs To determine whether other 5' nucleases in other DNAPs would be suitable for the present invention, an array of enzymes, several of which were reported in the literature to be free of appaeii 5' nuclease activity, were examined. The ability of these other enzymes to cleave nucleic acids in a structure-specific manner was tested using the hairpin substrate shown in Fig. 6 under conditions reported to be optimal for synthesis by each enzyme.

DNAPEcl and DNAP Klenow were obtained from Promega Corporation; the DNAP of Pyrococcus furious Bargseid et al., Strategies 4:34 [1991]) was from Strategene; the DNAP of Thermococcus litoralis VentTM(exo-), Perler et al., Proc. Natl. Acad. Sci., USA 89:5577 [1992]) was from New England Biolabs; the DNAP of Thermusflavus ("Tfl", Kaledin et al.. Biokhimiva 46:1576 [1981]) was from Epicentre Technologies; and the DNAP of Thermus thermophilus Carballeira et al., Biotechniques 9:276 [1990]; Myers et al., Biochem.. 30:7661 [1991]) was from U.S. Biochemical.

In this Example, 0.5 units of each DNA polymerase was assayed in a 20 pl reaction, using either the buffers supplied by the manufacturers for the primer-dependent reactions, or mM Tris-Cl, pH 8.5. 1.5 mM MgCI 2 and 20mM KCI. Reaction mixtures were at held 72 0 C before the addition of enzyme.

Fig. 11 is an autoradiogram recording the results of these tests. Fig. 11 A demonstrates reactions of endonucleases of DNAPs of several thermophilic bacteria. The reactions were incubated at 55°C for 10 minutes in the presence of primer or at 72 0 C for 30 minutes in the absence of primer, and the products were resolved by denaturing polyacrylamide gel electrophoresis. The lengths of the products, in nucleotides, are indicated. Fig. 1B demonstrates endonucleolytic cleavage by the 5' nuclease of DNAPEcl. The DNAPEcl and DNAP Klenow reactions were incubated for 5 minutes at 37°C. Note the light band of 25 cleavage products of 25 and 11 nucleotides in the DNAPEcl lanes (made in the presence and absence of primer, respectively). Fig. 7B also demonstrates DNAPTaq reactions in the presence or absence of primer. These reactions were run in 50 mM and 20 mM KC1, respectively, and were incubated at 55°C for 10 minutes.

Referring to Fig. 11A, DNAPs from the eubacteria Thermus thermophilus and Thermus flavus cleave the substrate at the same place as DNAPTaq, both in the presence and absence of primer. In contrast, DNAPs from the archaebacteria Pyrococcus furiosus and Thermococcus litoralis are unable to cleave the substrates endonucleolytically. The DNAPs from Pyrococcus furious and Thermococcus litoralis share little sequence homology with -98 eubacterial enzymes (Ito et al., Nucl. Acids Res., 19:4045 [1991]; Mathur et al.. Nucl. Acids Res., 19:6952 [1991]; See also Perler el Referring to Fig. 11B, DNAPEcl also cleaves the substrate, but the resulting cleavage products are difficult to detect unless the 3' exonuciease is inhibited. The amino acid sequences of the 5' nuclease domains of DNAPEcl and DNAPTaq are about 38% homologous (Gelfand, supra).

The 5' nuclease domain of DNAPTaq also shares about 19% homology with the exonuclease encoded by gene 6 of bacteriophage T7 (Dunn et al., J. Mol. Biol. 166:477 [1983]). This nuclease, which is not covalently attached to a DNAP polymerization domain, is also able to cleave DNA endonucleolytically, at a site similar or identical to the site that is cut by the 5' nucleases described above, in the absence of added primers.

C. Transcleavage The ability of a 5' nuclease to be directed to cleave efficiently at any specific sequence was demonstrated in the following experiment. A partially complementary oligonucleotide 15 termed a "pilot oligonucleotide" was hybridized to sequences at the desired point of cleavage.

The non-complementary part of the pilot oligonucleotide provided a structure analogous to the 3' arm of the template (See, Fig. whereas the 5' region of the substrate strand became the 5' arm. A primer was provided by designing the 3' region of the pilot so that it would fold .on itself creating a short hairpin with a stabilizing tetra-loop (Antao et al., Nucl. Acids Res., 19:5901 [1991]). Two pilot oligonucleotides are shown in Fig. 12A. Oligonucleotides 19-12 (SEQ ID NO:18), 30-12 (SEQ ID NO:19) and 30-0 (SEQ ID NO:20) are 31, 42 or nucleotides long, respectively. However, oligonucleotides 19-12 (SEQ ID NO:18) and 34-19 (SEQ ID NO:19) have only 19 and 30 nucleotides, respectively, that are complementary to ~different sequences in the substrate strand. The pilot oligonucleotides are calculated to melt off their complements at about 50 0 C (19-12) and about 75°C (30-12). Both pilots have 12 nucleotides at their 3' ends, which act as 3' arms with base-paired primers attached.

To demonstrate that cleavage could be directed by a pilot oligonucleotide, we incubated a single-stranded target DNA with DNAPTaq in the presence of two potential pilot oligonucleotides. The transcleavage reactions, where the target and pilot nucleic acids are not covalently linked, includes 0.01 pmoles of single end-labeled substrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotide in a volume of 20 tl of the same buffers.

These components were combined during a one minute incubation at 95 0 C, to denature the PCR-generated double-stranded substrate DNA, and the temperatures of the reactions were -99then reduced to their final incubation temperatures. Oligonucleotides 30-12 and 19-12 can hybridize to regions of the substrate DNAs that are 85 and 27 nucleotides from the 5' end of the targeted strand.

fig. 21 shows te compileit 206-mer sequence (SEQ 1I NO:32). The 206-mer was generated by PCR The MI3/pUC 24-mer reverse sequencing primer and the M13/pUC sequencing primer from New England Biolabs (catalogue nos. 1233 and 1224 respectively) were used (50 pmoles each) with the pGEM3z(f+) plasmid vector-(Promega Corp.) as template (10 ng) containing the target sequences. The conditions for PCR were as follows: 50 pM of each dNTP and 2.5 units of Taq DNA polymerase in 100 pl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCI 2 50 mM KCI with 0.05% Tween-20 and 0.05% Reactions were cycled 35 times through 95°C for 45 seconds, 63 0 C for 45 seconds, then 72°C for 75 seconds. After cycling, reactions were finished off with an incubation at 72 0 C for minutes. The resulting fragment was purified by electrophoresis through a 6% polyacrylamide gel (29:1 cross link) in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, visualized by ethidium bromide staining or autoradiography, excised from the gel.

eluted by passive diffusion, and concentrated by ethanol precipitation.

SCleavage of the substrate DNA occurred in the presence of the pilot oligonucleotide 19-12 at 50 0 C (Fig. 12B. lanes I and 7) but not at 75 0 C (lanes 4 and 10). In the presence of oligonucleotide 30-12 cleavage was observed at both temperatures. Cleavage did not occur in the absence of added oligonucleotides (lanes 3, 6 and 12) or at about 80 0 C even though at 50 0 C adventitious structures in the substrate allowed primer-independent cleavage in the absence of KCI (Fig. 12B, lane A non-specific oligonucleotide with no complementarity to the substrate DNA did not direct cleavage at 50 0 C, either in the absence or presence of mM KCI (lanes 13 and 14). Thus, the specificity of the cleavage reactions can be controlled 25 by the extent of complementarity to the substrate and by the conditions of incubation.

D. Cleavage Of RNA SAn shortened RNA version of the sequence used in the transcleavage experiments discussed above was tested for its ability to serve as a substrate in the reaction. The RNA is cleaved at the expected place, in a reaction that is dependent upon the presence of the pilot oligonucleotide. The RNA substrate, made by T7 RNA polymerase in the presence of [a- "P]UTP. corresponds to a truncated version of the DNA substrate used in Fig. 12B. Reaction conditions were similar to those in used for the DNA substrates described above, with 50 mM 100 KCI; incubation was for 40 minutes at 55 0 C. The pilot oligonucleotide used is termed 30-0 (SEQ ID NO:20) and is shown in Fig. 13A.

The results of the cleavage reaction is shown in Fig. 13B. The reaction was run either in t:h presence cu absence of DNAPTaq or pilot oligonucleotide as indicated in Fig. 13B.

Strikingly, in the case of RNA cleavage, a 3' arm is not required for the pilot oligonucleotide. It is very unlikely that this cleavage is due to previously described RNaseH, which would be expected to cut the RNA in several places along the 30 base-paif long RNA- DNA duplex. The 5' nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNA at a single site near the 5' end of the heteroduplexed region.

It is surprising that an oligonucleotide lacking a 3' arm is able to act as a pilot in directing efficient cleavage of an RNA target because such oligonucleotides are unable to direct efficient cleavage of DNA targets using native DNAPs. However, some 5' nucleases of the present invention (for example, clones E, F and G of Fig. 4) can cleave DNA in the absence of a 3' arm. In other words, a non-extendable cleavage structure is not required for specific cleavage with some 5' nucleases of the present invention derived from thermostable DNA polymerases.

We tested whether cleavage of an RNA template by DNAPTaq in the presence of a fully complementary primer could help explain why DNAPTaq is unable to extend a DNA oligonucleotide on an RNA template, in a reaction resembling that of reverse transcriptase.

Another thermophilic DNAP, DNAPTth, is able to use RNA as a template, but only in the presence of so we predicted that this enzyme would not cleave RNA in the presence of this cation. Accordingly, we incubated an RNA molecule with an appropriate pilot oligonucleotide in the presence of DNAPTaq or DNAPTth, in buffer containing either Mg++ or As expected, both enzymes cleaved the RNA in the presence of However, 25 DNAPTaq, but not DNAPTth, degraded the RNA in the presence of We conclude that the 5' nuclease activities of many DNAPs may contribute to their inability to use RNA as templates.

101 EXAMPLE 2 Generation Of 5' Nucleases From Thermostable DNA Polymerases Thermostable DNA polymerases were generated which have reduced synthetic activity.

an activity that is an undesirable side-reaction during DNA cleavage in the detection assay of the invention, yet have maintained thermostable nuclease activity. The result is a thermostable polymerase which cleaves nucleic acids DNA with extreme specificity.

Type A DNA polymerases from eubacteria of the genus Thermus share extensive protein sequence identity (90% in the polymerization domain, using the Lipman-Pearson method in the DNA analysis software from DNAStar, WI) and behave similarly in both polymerization and nuclease assays. Therefore, we have used the genes for the DNA polymerase of Thermus aquaricus (DNAPTaq) and Thermus flavus (DNAPTfl) as representatives of this class. Polymerase genes from other eubacterial organisms, such as Thermus thermophilus. Thermus sp., Thermooga maritima, Thermosipho africanus and Bacillus stearothermophilus are equally suitable. The DNA polymerases from these thermophilic organisms are capable of surviving and performing at elevated temperatures, and can thus be used in reactions in which temperature is used as a selection against non-specific hybridization of nucleic acid strands.

The restriction sites used for deletion mutagenesis, described below, were chosen for convenience. Different sites situated with similar convenience are available in the Thermus thermophilus gene and can be used to make similar constructs with other Type A polymerase genes from related organisms.

A. Creation Of 5' Nuclease Constructs 1. Modified DNAPTaq Genes 25 The first step was to place a modified gene for the Taq DNA polymerase on a plasmid under control of an inducible promoter. The modified Taq polymerase gene was isolated as follows: The Taq DNA polymerase gene was amplified by polymerase chain reaction from genomic DNA from Thermus aquaticus, strain YT-1 (Lawyer et al., supra), using as primers the oligonucleotides described in SEQ ID NOS:13-14. The resulting fragment of DNA has a recognition sequence for the restriction endonuclease EcoRI at the 5' end of the coding sequence and a BglII sequence at the 3' end. Cleavage with BglII leaves a 5' overhang or "sticky end" that is compatible with the end generated by BamHI. The PCR-amplified DNA was digested with EcoRJ and BamHI. The 2512 bp fragment containing the coding region for 102 the polymerase gene was gel purified and then ligated into a plasmid which contains an inducible promoter.

In one embodiment of the invention, the pTTQ18 vector, which contains the hybrid trp-lac (tac) promoter, was used Stark, Gene 5:255 (1987)] and shown in Fig. 14.

The tac promoter is under the control of the E. coli lac repressor. Repression allows the synthesis of the gene product to be suppressed until the desired level of bacterial growth has been achieved, at which point repression is removed by addition of a specific inducer, isopropyl-P-D-thiogalactopyranoside (IPTG). Such a system allows the expression of foreign proteins that may slow or prevent growth of transformants.

Bacterial promoters, such as tac, may not be adequately suppressed when they are present on a multiple copy plasmid. If a highly toxic protein is placed under control of such a promoter, the small amount of expression leaking through can be harmful to the bacteria.

In another embodiment of the invention, another option for repressing synthesis of a cloned gene product was used. The non-bacterial promoter, from bacteriophage T7, found in the plasmid vector series pET-3 was used to express the cloned mutant Taq polymerase genes [Fig. 15; Studier and Moffatt, J. Mol. Biol. 189:113 (1986)]. This promoter initiates transcription only by T7 RNA polymerase. In a suitable strain, such as BL21(DE3)pLYS, the gene for this RNA polymerase is carried on the bacterial genome under control of the lac operator. This arrangement has the advantage that expression of the multiple copy gene (on the plasmid) is completely dependent on the expression of T7 RNA polymerase, which is easily suppressed because it is present in a single copy.

*'For ligation into the pTTQI8 vector (Fig. 14), the PCR product DNA containing the Taq polymerase coding region (mutTaq, clone 4B, SEQ ID NO:21) was digested with EcoRI and BglII and this fragment was ligated under standard "sticky end" conditions [Sambrook et 25 al. Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1.63- 1.69 (1989)] into the EcoRI and BamHI sites of the plasmid vector pTTQI8. Expression of this construct yields a translational fusion product in which the first two residues of the native protein (Met-Arg) are replaced by three from the vector (Met-Asn-Ser), but the remainder of the natural protein would not change. The construct was transformed into the JM109 strain of E. coli and the transformants were plated under incompletely repressing conditions that do not permit growth of bacteria expressing the native protein. These plating conditions allow the isolation of genes containing pre-existing mutations, such as those that result from the infidelity of Taq polymerase during the amplification process.

103 Using this amplification/selection protocol, we isolated a clone (depicted in Fig. 4B) containing a mutated Taq polymerase gene (mutTaq, clone 4B). The mutant was first detected by its phenotype, in which temperature-stable 5' nuclease activity in a crude cell extract was normal, hbut po!ymer.zao ativity was aiiuost absent (approximately less than 1% of wild type Taq polymerase activity).

DNA sequence analysis of the recombinant gene showed that it had changes in the polymerase domain resulting in two amino acid substitutions: an A to G change at nucleotide position 1394 causes a Glu to Gly change at amino acid position 465 (numbered according to the natural nucleic and amino acid sequences, SEQ ID NOS:l and 4) and another A to G change at nucleotide position 2260 causes a Gin to Arg change at amino acid position 754.

Because the Gln to Gly mutation is at a nonconserved position and because the Glu to Arg mutation alters an amino acid that is conserved in virtually all of the known Type A polymerases, this latter mutation is most likely the one responsible for curtailing the synthesis activity of this protein. The nucleotide sequence for the Fig. 4B construct is given in SEQ ID NO:21. The enzyme encoded by this sequence is referred to as Cleavase® A/G.

SSubsequent derivatives of DNAPTaq constructs were made from the mutTaq gene, thus, they all bear these amino acid substitutions in addition to their other alterations, unless these particular regions were deleted. These mutated sites are indicated by black boxes at these locations in the diagrams in Fig. 4. In Fig. 4, the designation Exo" is used to indicate the location of the 3' exonuclease activity associated with Type A polymerases which is not present in DNAPTaq. All constructs except the genes shown in Figs. 4E, F and G were made in the pTTQ18 vector.

The cloning vector used for the genes in Figs. 4E and F was from the commercially available pET-3 series, described above. Though this vector series has only a BamHI site for 25 cloning downstream of the T7 promoter, the series contains variants that allow cloning into any of the three reading frames. For cloning of the PCR product described above, the variant called pET-3c was used (Fig 15). The vector was digested with BamHI, dephosphorylated with calf intestinal phosphatase, and the sticky ends were filled in using the Klenow fragment of DNAPEcl and dNTPs. The gene for the mutant Taq DNAP shown in Fig. 4B (mutTaq, clone 4B) was released from pTTQ18 by digestion with EcoRI and Sail, and the "sticky ends" were filled in as was done with the vector. The fragment was ligated to the vector under standard blunt-end conditions (Sambrook et al., Molecular Cloning, supra), the construct was transformed into the BL21(DE3)pLYS strain of E. coli, and isolates were 104screened to identify those that were ligated with the gene in the proper orientation relative to the promoter. This construction yields another translational fusion product, in which the first two amino acids of DNAPTaq (Met-Arg) are replaced by 13 from the vector plus two from the PCR primer (Met-Aia-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ ID NO:29).

Our goal was to generate enzymes that lacked the ability to synthesize DNA, but retained the ability to cleave nucleic acids with a 5' nuclease activity. The act of primed, templated synthesis of DNA is actually a coordinated series of events, so it is possible to disable DNA synthesis by disrupting one event while not affecting the others. These steps include, but are not limited to, primer recognition and binding, dNTP binding and catalysis of the inter-nucleotide phosphodiester bond. Some of the amino acids in the polymerization domain of DNAPEcI have been linked to these functions, but the precise mechanisms are as yet poorly defined.

One way of destroying the polymerizing ability of a DNA polymerase is to delete all or part of the gene segment that encodes that domain for the protein, or to otherwise render the gene incapable of making a complete polymerization domain. Individual mutant enzymes may differ from each other in stability and solubility both inside and outside cells. For instance, in contrast to the 5' nuclease domain of DNAPEcI, which can be released in an active form from the polymerization domain by gentle proteolysis (Setlow and Kornberg, J.

Biol. Chem., 247:232 [1972]), the Thermus nuclease domain, when treated similarly, becomes less soluble and the cleavage activity is often lost.

Using the mutant gene shown in Fig. 4B as starting material, several deletion constructs were created. All cloning technologies were standard (Sambrook et al., supra) and are summarized briefly, as follows: 25 Fig. 4C: The mutTaq construct was digested with PstI, which cuts once within the polymerase coding region, as indicated, and cuts immediately downstream of the gene in the multiple cloning site of the vector. After release of the fragment between these two sites, the vector was re-ligated, creating an 894-nucleotide deletion, and bringing into frame a stop codon 40 nucleotides downstream of the junction. The nucleotide sequence of this nuclease (clone 4C) is given in SEQ ID NO:9.

Fig. 4D: The mutTaq construct was digested with Nhel, which cuts once in the gene at position 2047. The resulting four-nucleotide 5' overhanging ends were filled in, as described above, and the blunt ends were re-ligated. The resulting four-nucleotide insertion 105 changes the reading frame and causes termination of translation ten amino acids downstream of the mutation. The nucleotide sequence of this 5' nuclease (clone 4D) is given in SEQ ID Fig. 4E: The entire mutTaa gene waz rut from pTTQIS using Eco1 and Sl andU cloned into pET-3c, as described above. This clone was digested with BstXI and Xcml, at unique sites that are situated as shown in Fig. 4E. The DNA was treated with the Klenow fragment of DNAPEcl and dNTPs, which resulted in the 3' overhangs of both sites being trimmed to blunt ends. These blunt ends were ligated together, resulting in an out-of-frame deletion of 1540 nucleotides. An in-frame termination codon occurs 18 triplets past the junction site. The nucleotide sequence of this 5' nuclease (clone 4E) is given in SEQ ID NO:11, with the appropriate leader sequence given in SEQ ID NO:30. It is also referred to as Cleavase® BX.

Fig. 4F: The entire mutTaq gene was cut from pTTQ18 using EcoRI and Sal and cloned into pET-3c, as described above. This clone was digested with BstXI and BamHI, at unique sites that are situated as shown in the diagram. The DNA was treated with the Klenow fragment of DNAPEcl and dNTPs, which resulted in the 3' overhang of the BsIXI site being trimmed to a blunt end, while the 5' overhang of the BamHI site was filled in to make a blunt end. These ends were ligated together, resulting in an in-frame deletion of 903 nucleotides. The nucleotide sequence of the 5' nuclease (clone 4F) is given in SEQ ID 20 NO:12. It is also referred to as Cleavase® BB.

Fig.4G: This polymerase is a variant of that shown in Fig. 4E. It was cloned in the plasmid vector pET-21 (Novagen). The non-bacterial promoter from bacteriophage T7, found in this vector, initiates transcription only by T7 RNA polymerase. See Studier and Moffatt, supra. In a suitable strain, such as (DES)pLYS, the gene for this RNA polymerase is carried 25 on the bacterial genome under control of the lac operator. This arrangement has the advantage that expression of the multiple copy gene (on the plasmid) is completely dependent on the expression of T7 RNA polymerase, which is easily suppressed because it is present in a single copy. Because the expression of these mutant genes is under this tightly controlled promoter, potential problems of toxicity of the expressed proteins to the host cells are less of a concern.

The pET-21 vector also features a "His*Tag", a stretch of six consecutive histidine residues that are added on the carboxy terminus of the expressed proteins. The resulting proteins can then be purified in a single step by metal chelation chromatography, using a 106commercially available (Novagen) column resin with immobilized Ni" ions. The 2.5 ml columns are reusable, and can bind up to 20 mg of the target protein under native or denaturing (guanidine*HCI or urea) conditions.

E. coli (DES)pLYS cells are transformed with the constructs described above using standard transformation techniques, and used to inoculate a standard growth medium Luria-Bertani broth). Production of T7 RNA polymerase is induced during log phase growth by addition of IPTG and incubated for a further 12 to 17 hours. Aliquots of culture are removed both before and after induction and the proteins are examined by SDS-PAGE.

Staining with Coomassie Blue allows visualization of the foreign proteins if they account for about 3-5% of the cellular protein and do not co-migrate with any of the major protein bands.

Proteins that co-migrate with major host protein must be expressed as more than 10% of the total protein to be seen at this stage of analysis.

Some mutant proteins are sequestered by the cells into inclusion bodies. These are granules that form in the cytoplasm when bacteria are made to express high levels of a foreign protein, and they can be purified from a crude lysate, and analyzed by SDS-PAGE to determine their protein content. If the cloned protein is found in the inclusion bodies, it must be released to assay the cleavage and polymerase activities. Different methods of solubilization may be appropriate for different proteins, and a variety of methods are known.

See Builder Ogez, U.S. Patent No. 4,511,502 (1985); Olson, U.S. Patent No.

4,518,526 (1985); Olson Pai, U.S. Patent No. 4,511,503 (1985); Jones et al., U.S. Patent No. 4,512,922 (1985), all of which are hereby incorporated by reference.

The solubilized protein is then purified on the Ni" column as described above, following the manufacturers instructions (Novagen). The washed proteins are eluted from the column by a combination of imidazole competitor (1 M) and high salt (0.5 M NaCI), and 25 dialyzed to exchange the buffer and to allow denature proteins to refold. Typical recoveries result in approximately 20 pg of specific protein per ml of starting culture. The DNAP mutant is referred to as the Cleavase® BN nuclease and the sequence is given in SEQ ID NO:31 (the amino acid sequence of the Cleavase® BN nuclease is obtained by translating the DNA sequence of SEQ ID NO:31).

107- 2. Modified DNAPTfl Gene The DNA polymerase gene of Thermusflavus was isolated from the "T flavus" AT-62 strain obtained from the ATCC (ATCC #33923). This strain has a different restriction map then does the T. flavus strain used to generate the sequence p ublshed by Akh.i-.-etzao and Vakhitov, supra. The published sequence is listed as SEQ ID NO:2. No sequence data has been published for the DNA polymerase gene from the AT-62 strain of T. flavus.

Genomic DNA from T. flavus was amplified using the same primers used to amplify the T aquaticus DNA polymerase gene (SEQ ID NOS:13-14). The approximately 2500 base pair PCR fragment was digested with EcoRI and BamHI. The over-hanging ends were made blunt with the Klenow fragment of DNAPEcl and dNTPs. The resulting approximately 1800 base pair fragment containing the coding region for the N-terminus was ligated into pET-3c, as described above. This construct, clone 5B, is depicted in Fig. 5B. The wild type T flavus DNA polymerase gene is depicted in Fig. 5A. The 5B clone has the same leader amino acids as do the DNAPTaq clones 4E and F which were cloned into pET-3c; it is not known precisely where translation termination occurs, but the vector has a strong transcription termination signal immediately downstream of the cloning site.

°*eeo SB. Growth And Induction Of Transformed Cells S* Bacterial cells were transformed with the constructs described above using standard 20 transformation techniques and used to inoculate 2 mis of a standard growth medium Luria-Bertani broth). The resulting cultures were incubated as appropriate for the particular strain used, and induced if required for a particular expression system. For all of the constructs depicted in Figs. 4 and 5, the cultures were grown to an optical density (at 600nm wavelength) of 0.5 OD.

25 To induce expression of the cloned genes, the cultures were brought to a final concentration of 0.4 mM IPTG and the incubations were continued for 12 to 17 hours. 50 ul aliquots of each culture were removed both before and after induction and were combined with 20 ptl of a standard gel loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue (Sambrook et al., supra) allows visualization of the foreign proteins if they account for about 3-5% of the cellular protein and do not co-migrate with any of the major E. coli protein bands. Proteins that do co-migrate with a major host protein must be expressed as more than 10% of the total protein to be seen at this stage of analysis.

108 C. Heat Lysis And Fractionation Expressed thermostable proteins, the 5' nucleases, were isolated by heating crude bacterial cell extracts to cause denaturation and precipitation of the less stable E. coli proteins.

The precipitated E. coli proteins were then, along with other cell debris, removed by centrifugation. 1.7 mis of the culture were pelleted by microcentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. After removal of the supernatant, the cells were resuspended in 400 1l of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, I mM EDTA), re-centrifuged, then resuspended in 80 /l of buffer A with 4mg/ml lysozyme. The cells were incubated at room temperature for 15 minutes, then combined with 80 pl of buffer B (10 mM Tris-HC1, pH 7.9, 50 mM KCI. 1 mM EDTA, 1 mM PMSF, 0.5% This mixture was incubated at 75°C for 1 hour to denature and precipitate the host proteins. This cell extract was centrifuged at 14,000 rpm for 15 minutes at 4 0 C, and the supernatant was transferred to a fresh tube. An aliquot of 0.5 to 1 /l of this supernatant was used directly in each test reaction, and the protein content of the extract was determined by subjecting 7 xl to electrophoretic analysis, as above. The native recombinant Taq DNA polymerase [Englke, Anal. Biochem 191:396 (1990)], and the double point mutation protein shown in Fig. 4B are both soluble and active at this point.

The foreign protein may not be detected after the heat treatments due to sequestration of the foreign protein by the cells into inclusion bodies. These are granules that form in the cytoplasm when bacteria are made to express high levels of a foreign protein, and they can be purified from a crude lysate, and analyzed SDS PAGE to determine their protein content.

Many methods have been described in the literature, and one approach is described below.

D. Isolation And Solubilization Of Inclusion Bodies 25 A small culture was grown and induced as described above. A 1.7 ml aliquot was pelleted by brief centrifugation, and the bacterial cells were resuspended in 100 /l of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaC1). 2.5 /l of 20 mM PMSF were added for a final concentration of 0.5 mM, and lysozyme was added to a concentration of 1.0 mg/ml. The cells were incubated at room temperature for 20 minutes, deoxycholic acid was added to 1mg/ml (1 /l of 100 mg/ml solution), and the mixture was further incubated at 37 0 C for about 15 minutes or until viscous. DNAse I was added to 10 /g/ml and the mixture was incubated at room temperature for about 30 minutes or until it was no longer viscous.

109- From this mixture the inclusion bodies were collected by centrifugation at 14.000 rpm for 15 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in 100 pl of lysis buffer with 10mM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutes at room temperature, the inclusion bodies were pelleted as before, and the supernataiL was saved for later analysis. The inclusion bodies were resuspended in 50 p.1 of distilled water, and 5 pl was combined with SDS gel loading buffer (which dissolves the inclusion bodies) and analyzed electrophoretically, along with an aliquot of the supernatant.

If the cloned protein is found in the inclusion bodies, it may be released to assay the cleavage and polymerase activities and the method of solubilization must be compatible with the particular activity. Different methods of solubilization may be appropriate for different proteins, and a variety of methods are discussed in Molecular Cloning (Sambrook et al..

supra). The following is an adaptation we have used for several of our isolates.

Twenty pl of the inclusion body-water suspension were pelleted by centrifugation at 14,000 rpm for 4 minutes at room temperature. and the supernatant was discarded. To further wash the inclusion bodies, the pellet was resuspended in 20 pl of lysis buffer with 2M urea, and incubated at room temperature for one hour. The washed inclusion bodies were then resuspended in 2 pl of lysis buffer with 8M urea; the solution clarified visibly as the inclusion bodies dissolved. Undissolved debris was removed by centrifugation at 14,000 rpm for 4 minutes at room temperature, and the extract supernatant was transferred to a fresh tube.

20 To reduce the urea concentration, the extract was diluted into KH 2

PO

4 A fresh tube was prepared containing 180 tl of 50 mM KHPO 4 pH 9.5, 1 mM EDTA and 50 mM NaCI.

A 2 pI aliquot of the extract was added and vortexed briefly to mix. This step was repeated until all of the extract had been added for a total of 10 additions. The mixture was allowed to sit at room temperature for 15 minutes, during which time some precipitate often forms.

25 Precipitates were removed by centrifugation at 14,000 rpm, for 15 minutes at room temperature, and the supernatant was transferred to a fresh tube. To the 200 l1 of protein in the KH 2 PO, solution, 140-200 pl of saturated (NH 4 2

SO

4 were added, so that the resulting mixture was about 41% to 50% saturated (NH 4 2

SO

4 The mixture was chilled on ice for minutes to allow the protein to precipitate, and the protein was then collected by centrifugation at 14,000 rpm, for 4 minutes at room temperature. The supernatant was discarded, and the pellet was dissolved in 20 p Buffer C (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.5% PMSF, 25 mM KCI and 0.5 each of Tween-20 and Nonidet P 40). The protein solution was centrifuged again for 4 minutes to pellet insoluble materials, and the -110supernatant was removed to a fresh tube. The protein contents of extracts prepared in this manner were visualized by resolving 1-4 .l by SDS-PAGE; 0.5 to I p.l of extract was tested in the cleavage and polymerization assays as described.

E. Fruiein Analysis For Presence Of Nuclease And Synthetic Activity The 5' nucleases described above and shown in Figs. 4 and 5 were analyzed by the following methods.

1. Structure Specific Nuclease Assay A candidate modified polymerase is tested for 5' nuclease activity by examining its ability to catalyze structure-specific cleavages. By the term "cleavage structure" as used herein, is meant a nucleic acid structure which is a substrate for cleavage by the 5' nuclease activity of a DNAP.

The polymerase is exposed to test complexes that have the structures shown in Fig. 16.

Testing for 5' nuclease activity involves three reactions: 1) a primer-directed cleavage (Fig.

16B) is performed because it is relatively insensitive to variations in the salt concentration of .the reaction and can, therefore, be performed in whatever solute conditions the modified enzyme requires for activity; this is generally the same conditions preferred by unmodified polymerases; 2) a similar primer-directed cleavage is performed in a buffer which permits primer-independent cleavage, a low salt buffer, to demonstrate that the enzyme is viable under these conditions; and 3) a primer-independent cleavage (Fig. 16A) is performed in the same low salt buffer.

The bifurcated duplex is formed between a substrate strand and a template strand as shown in Fig. 16. By the term "substrate strand" as used herein, is meant that strand of nucleic acid in which the cleavage mediated by the 5' nuclease activity occurs. The substrate 25 strand is always depicted as the top strand in the bifurcated complex which serves as a substrate for 5' nuclease cleavage (Fig. 16). By the term "template strand" as used herein, is meant the strand of nucleic acid which is at least partially complementary to the substrate strand and which anneals to the substrate strand to form the cleavage structure. The template strand is always depicted as the bottom strand of the bifurcated cleavage structure (Fig. 16).

If a primer (a short oligonucleotide of 19 to 30 nucleotides in length) is added to the complex, as when primer-dependent cleavage is to be tested, it is designed to anneal to the 3' arm of the template strand (Fig. 16B). Such a primer would be extended along the template strand if the polymerase used in the reaction has synthetic activity.

111 The cleavage structure may be made as a single hairpin molecule, with the 3' end of the target and the 5' end of the pilot joined as a loop as shown in Fig. 16E. A primer oligonucleotide complementary to the 3' arm is also required for these tests so that the en.zyme's sensitivity t the preence of a primci maiy be iesLed.

Nucleic acids to be used to form test cleavage structures can be chemically synthesized, or can be generated by standard recombinant DNA techniques. By the latter method, the hairpin portion of the molecule can be created by inserting into a cloning vector duplicate copies of a short DNA segment, adjacent to each other but in opposing orientation.

The double-stranded fragment encompassing this inverted repeat, and including enough flanking sequence to give short (about 20 nucleotides) unpaired 5' and 3' arms, can then be released from the vector by restriction enzyme digestion, or by PCR performed with an enzyme lacking a 5' exonuclease the Stoffel fragment of Amplitaq T M DNA polymerase, VentTM DNA polymerase).

The test DNA can be labeled on either end, or internally, with either a radioisotope, or with a non-isotopic tag. Whether the hairpin DNA is a synthetic single strand or a cloned 6 S* double strand, the DNA is heated prior to use to melt all duplexes. When cooled on ice, the structure depicted in Fig. 16E is formed, and is stable for sufficient time to perform these assays.

To test for primer-directed cleavage (Reaction a detectable quantity of the test 20 molecule (typically 1-100 fmol of 2 P-labeled hairpin molecule) and a 10 to 100-fold molar excess of primer are placed in a buffer known to be compatible with the test enzyme. For Reaction 2, where primer-directed cleavage is performed under condition which allow primerindependent cleavage, the same quantities of molecules are placed in a solution that is the same as the buffer used in Reaction I regarding pH, enzyme stabilizers bovine serum 0 25 albumin, nonionic detergents, gelatin) and reducing agents dithiothreitol, 2-mercaptoethanol) but that replaces any monovalent cation salt with 20 mM KCI; 20 mM KCI is the demonstrated optimum for primer-independent cleavage. Buffers for enzymes, such as DNAPEcl, that usually operate in the absence of salt are not supplemented to achieve this concentration. To test for primer-independent cleavage (Reaction 3) the same quantity of the test molecule, but no primer, are combined under the same buffer conditions used for Reaction 2.

112- All three test reactions are then exposed to enough of the enzyme that the molar ratio of enzyme to test complex is approximately 1:1. The reactions are incubated at a range of temperatures up to, but not exceeding, the temperature allowed by either the enzyme stability or the coiumpiCA s taIjuty, whichever is lower, up to 8U"C for enzymes from thermophiles, for a time sufficient to allow cleavage (10 to 60 minutes). The products of Reactions 1, 2 and 3 are resolved by denaturing polyacrylamide gel electrophoresis, and visualized by autoradiography or by a comparable method appropriate to the labeling system used.

Additional labeling systems include chemiluminescence detection, silver or other stains, blotting and probing and the like. The presence of cleavage products is indicated by the presence of molecules which migrate at a lower molecular weight than does the uncleaved test structure. These cleavage products indicate that the candidate polymerase has structurespecific 5' nuclease activity.

To determine whether a modified DNA polymerase has substantially the same nuclease activity as that of the native DNA polymerase, the results of the above-described tests are compared with the results obtained from these tests performed with the native DNA polymerase. By "substantially the same 5' nuclease activity" we mean that the modified polymerase and the native polymerase will both cleave test molecules in the same manner. It is not necessary that the modified polymerase cleave at the same rate as the native DNA polymerase.

Some enzymes or enzyme preparations may have other associated or contaminating activities that may be functional under the cleavage conditions described above and that may interfere with 5' nuclease detection. Reaction conditions can be modified in consideration of these other activities, to avoid destruction of the substrate, or other masking of the 5' nuclease cleavage and its products. For example, the DNA polymerase I of E. coli (Pol in addition 25 to its polymerase and 5' nuclease activities, has a 3' exonuclease that can degrade DNA in a 3' to 5' direction. Consequently, when the molecule in Fig. 16E is exposed to this polymerase under the conditions described above, the 3' exonuclease quickly removes the unpaired 3' arm, destroying the bifurcated structure required of a substrate for the exonuclease cleavage and no cleavage is detected. The true ability of Pol I to cleave the o*e 30 structure can be revealed if the 3' exonuclease is inhibited by a change of conditions pH), mutation, or by addition of a competitor for the activity. Addition of 500 pmoles of a single-stranded competitor oligonucleotide, unrelated to the Fig. 16E structure, to the cleavage reaction with Pol I effectively inhibits the digestion of the 3' arm of the Fig. 16E structure 113without interfering with the 5' exonuclease release of the 5' arm. The concentration of the competitor is not critical, but should be high enough to occupy the 3' exonuclease for the duration of the reaction.

Similar destruction of the test molecu!e may be caused by contaniiainm in the candidate polymerase preparation. Several sets of the structure specific nuclease reactions may be performed to determine the purity of the candidate nuclease and to find the window between under and over exposure of the test molecule to the polymerase preparation being investigated.

The above described modified polymerases were tested for 5' nuclease activity as follows: Reaction 1 was performed in a buffer of 10 mM Tris-C1, pH 8.5 at 20 0 C, 1.5 mM MgC1, and 50 mM KCI and in Reaction 2 the KCI concentration was reduced to 20 mM. In Reactions 1 and 2, 10 fmoles of the test substrate molecule shown in Fig. 16E were combined with 1 pmole of the indicated primer and 0.5 to 1.0 l of extract containing the modified polymerase (prepared as described above). This mixture was then incubated for 10 minutes at 55°C. For all of the mutant polymerases tested these conditions were sufficient to give complete cleavage. When the molecule shown in Fig. 16E was labeled at the 5' end, the released 5' fragment, 25 nucleotides long, was conveniently resolved on a polyacrylamide gel (19:1 cross-linked) with 7 M urea in a buffer containing 45 mM Trisborate pH 8.3, 1.4 mM EDTA. Clones 4C-F and 5B exhibited structure-specific cleavage comparable to that of the unmodified DNA polymerase. Additionally, clones 4E, 4F and 4G have the added ability to cleave DNA in the absence of a 3' arm as discussed above.

Representative cleavage reactions are shown in Fig. 17.

For the reactions shown in Fig. 17, the mutant polymerase clones 4E (Taq mutant) and (Tfl mutant) were examined for their ability to cleave the hairpin substrate molecule 25 shown in Fig. 16E. The substrate molecule was labeled at the 5' terminus with 32 P. fmoles of heat-denatured, end-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1) or 0.5 pl of 4e or 5b extract (Fig. 17, lanes 2-7, extract was prepared as described above) were mixed together in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCI and 1.5 mM MgCl 2 The final reaction volume was 10 pl. Reactions shown in lanes 4 and 7 contain in 30 addition 50 iM of each dNTP. Reactions shown in lanes 3, 4, 6 and 7 contain 0.2 pM of the primer oligonucleotide (complementary to the 3' arm of the substrate and shown in Fig. 16E).

Reactions were incubated at 550 C for 4 minutes. Reactions were stopped by the addition of 8 pl of 95% formamide containing 20 mM EDTA and 0.05% marker dyes per 10 pl reaction *e e*M 114volume. Samples were then applied to 12% denaturing acrylamide gels. Following electrophoresis, the gels were autoradiographed. Fig. 17 shows that clones 4E and 5B exhibit cleavage activity similar to that of the native DNAPTaq. Note that some cleavage occurs in these reactions in the absence of the primer. When long hairpin structure, such as the one used here (Fig. 16E), are used in cleavage reactions performed in buffers containing 50 mM KCI a low level of primer-independent cleavage is seen. Higher concentrations of KCI suppress, but do not eliminate, this primer-independent cleavage under these conditions.

2. Assay For Synthetic Activity The ability of the modified enzyme or proteolytic fragments is assayed by adding the modified enzyme to an assay system in which a primer is annealed to a template and DNA synthesis is catalyzed by the added enzyme. Many standard laboratory techniques employ such an assay. For example, nick translation and enzymatic sequencing involve extension of a primer along a DNA template by a polymerase molecule.

In a preferred assay for determining the synthetic activity of a modified enzyme an oligonucleotide primer is annealed to a single-stranded DNA template, bacteriophage M13 DNA, and the primer/template duplex is incubated in the presence of the modified polymerase in question, deoxynucleoside triphosphates (dNTPs) and the buffer and salts known to be appropriate for the unmodified or native enzyme. Detection of either primer extension (by denaturing gel electrophoresis) or dNTP incorporation (by acid precipitation or chromatography) is indicative of an active polymerase. A label, either isotopic or nonisotopic, is preferably included on either the primer or as a dNTP to facilitate detection of polymerization products. Synthetic activity is quantified as the amount of free nucleotide •incorporated into the growing DNA chain and is expressed as amount incorporated per unit of time under specific reaction conditions.

25 Representative results of an assay for synthetic activity is shown in Fig. 18. The synthetic activity of the mutant DNAPTaq clones 4B-F was tested as follows: A master mixture of the following buffer was made: 1.2X PCR buffer (IX PCR buffer contains mM KC1, 1.5 mM MgCl 2 10 mM Tris-Cl, pH 8.5 and 0.05% each Tween 20 and Nonidet 50 pM each of dGTP, dATP and dTTP, 5 pM dCTP and 0.125 pM a- 3 2 P-dCTP at 600 30 Ci/mmol. Before adjusting this mixture to its final volume, it was divided into two equal aliquots. One received distilled water up to a volume of 50 pl to give the concentrations above. The other received 5 pg of single-stranded M13mpl8 DNA (approximately 2.5 pmol or 0.05 jiM final concentration) and 250 pmol of M13 sequencing primer (5 pM final *o 115 concentration) and distilled water to a final volume of 50 pl. Each cocktail was warmed to 0 C for 5 minutes and then cooled to room temperature. This allowed the primers to anneal to the DNA in the DNA-containing mixtures.

For each assay, 4 Vl of the cocktail with the DNA was combined 'ith I il of Uih mutant polymerase, prepared as described, or 1 unit of DNAPTaq (Perkin Elmer) in 1 pa of dH0O. A "no DNA" control was done in the presence of the DNAPTaq (Fig. 18, lane and a "no enzyme" control was done using water in place of the enzyme (lane Each reaction was mixed, then incubated at room temperature (approx. 22 0 C) for 5 minutes, then at 55 0

C

for 2 minutes, then at 72 0 C for 2 minutes. This step incubation was done to detect polymerization in any mutants that might have optimal temperatures lower than 72 0 C. After the final incubation, the tubes were spun briefly to collect any condensation and were placed on ice. One Vil of each reaction was spotted at an origin 1.5 cm from the bottom edge of a polyethyleneimine (PEI) cellulose thin layer chromatography plate and allowed to dry. The chromatography plate was run in 0.75 M NaH,PO,, pH 3.5, until the buffer front had run approximately 9 cm from the origin. The plate was dried, wrapped in plastic wrap, marked with luminescent ink, and exposed to X-ray film. Incorporation was detected as counts that stuck where originally spotted, while the unincorporated nucleotides were carried by the salt solution from the origin.

Comparison of the locations of the counts with the two control lanes confirmed the lack of polymerization activity in the mutant preparations. Among the modified DNAPTaq clones, only clone 4B retains any residual synthetic activity as shown in Fig. 18.

EXAMPLE 3 Nucleases Derived From Thermostable DNA 2 5 Polymerases Can Cleave Short Hairpin Structures With Specificity The ability of the 5' nucleases to cleave hairpin structures to generate a cleaved hairpin structure suitable as a detection molecule was examined. The structure and sequence S of the hairpin test molecule is shown in Fig. 19A (SEQ ID NO:15). The oligonucleotide (labeled "primer" in Fig. 19A, SEQ ID NO:22) is shown annealed to its complementary 30 sequence on the 3' arm of the hairpin test molecule. The hairpin test molecule was single-end labeled with 2 P using a labeled T7 promoter primer in a polymerase chain reaction. The label is present on the 5' arm of the hairpin test molecule and is represented by the star in Fig. 19A.

116- The cleavage reaction was performed by adding 10 fmoles of heat-denatured, endlabeled hairpin test molecule, 0.2uM of the primer oligonucleotide (complementary to the 3' arm of the hairpin), 50 pM of each dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or pi of extract containing a 5' nuclease (prepared as described above) in a total volume of 10 pl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCI and 1.5 mM MgCl,. Reactions shown in lanes 3, 5 and 7 were run in the absence of dNTPs.

Reactions were incubated at 550 C for 4 minutes. Reactions were stopped at 550 C by the addition of 8 pl of 95% formamide with 20 mM EDTA and 0.05% marker dyes per 10 pl reaction volume. Samples were not heated before loading onto denaturing polyacrylamide gels (10% polyacrylamide, 19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3, 2.8 mM EDTA). The samples were not heated to allow for the resolution of single-stranded and reduplexed uncleaved hairpin molecules.

Fig. 19B shows that altered polymerases lacking any detectable synthetic activity cleave a hairpin structure when an oligonucleotide is annealed to the single-stranded 3' arm of the hairpin to yield a single species of cleaved product (Fig. 19B, lanes 3 and nucleases. such as clone 4D, shown in lanes 3 and 4, produce a single cleaved product even in the presence of dNTPs. 5' nucleases which retain a residual amount of synthetic activity (less than 1% of wild type activity) produce multiple cleavage products as the polymerase can extend the oligonucleotide annealed to the 3' arm of the hairpin thereby moving the site of cleavage (clone 4B, lanes 5 and Native DNAPTaq produces even more species of cleavage products than do mutant polymerases retaining residual synthetic activity and additionally converts the hairpin structure to a double-stranded form in the presence of dNTPs due to the high level of synthetic activity in the native polymerase (Fig. 19B, lane 8).

25 EXAMPLE 4 Test Of The Trigger/Detection Assay To test the ability of an oligonucleotide of the type released in the trigger reaction of the trigger/detection assay to be detected in the detection reaction of the assay, the two hairpin structures shown in Fig. 20A were synthesized using standard techniques. The two hairpins 30 are termed the A-hairpin (SEQ ID NO:23) and the T-hairpin (SEQ ID NO:24). The predicted sites of cleavage in the presence of the appropriate annealed primers are indicated by the arrows. The A- and T-hairpins were designed to prevent intra-strand mis-folding by omitting most of the T residues in the A-hairpin and omitting most of the A residues in the T-hairpin.

o* 117- To avoid mis-priming and slippage, the hairpins were designed with local variations in the sequence motifs spacing T residues one or two nucleotides apart or in pairs). The Aand T-hairpins can be annealed together to form a duplex which has appropriate ends for directional cloning in pIJC-tvnpe vpr.nrs; rPtrictin site e e loop egions of the f lA u up regin s of the duplex and can be used to elongate the stem regions if desired.

The sequence of the test trigger oligonucleotide is shown in Fig. 20B; this oligonucleotide is termed the alpha primer (SEQ ID NO:25). The alpha primer iscomplementary to the 3' arm of the T-hairpin as shown in Fig. 20A. When the alpha primer is annealed to the T-hairpin, a cleavage structure is formed that is recognized by thermostable DNA polymerases. Cleavage of the T-hairpin liberates the 5' single-stranded arm of the Thairpin, generating the tau primer (SEQ ID NO:26) and a cleaved T-hairpin (Fig. 20B; SEQ ID NO:27). The tau primer is complementary to the 3' arm of the A-hairpin as shown in Fig. 20A. Annealing of the tau primer to the A-hairpin generates another cleavage structure: cleavage of this second cleavage structure liberates the 5' single-stranded arm of the Ahairpin, generating another molecule of the alpha primer which then is annealed to another molecule of the T-hairpin. Thermocycling releases the primers so they can function in additional cleavage reactions. Multiple cycles of annealing and cleavage are carried out. The products of the cleavage reactions are primers and the shortened hairpin structures shown in Fig. 20C. The shortened or cleaved hairpin structures may be resolved from the uncleaved hairpins by electrophoresis on denaturing acrylamide gels.

The annealing and cleavage reactions are carried as follows: In a 50 pi reaction volume containing 10 mM Tris-C1, pH 8.5, 1.0 MgCI 2 75 mM KC1, I pmole of A-hairpin, I pmole T-hairpin, the alpha primer is added at equimolar amount relative to the hairpin structures (1 pmole) or at dilutions ranging from 10- to 10 6 -fold and 0.5 .1 of extract 25 containing a 5' nuclease (prepared as described above) are added. The predicted melting temperature for the alpha or trigger primer is 60 0 C in the above buffer. Annealing is performed just below this predicted melting temperature at 55 0 C. Using a Perkin Elmer DNA Thermal Cycler, the reactions are annealed at 55°C for 30 seconds. The temperature is then increased slowly over a five minute period to 72 0 C to allow for cleavage. After cleavage, the 30 reactions are rapidly brought to 55 0 C (1°C per second) to allow another cycle of annealing to occur. A range of cycles are performed (20, 40 and 60 cycles) and the reaction products are analyzed at each of these number of cycles. The number of cycles which indicates that the 118accumulation of cleaved hairpin products has not reached a plateau is then used for subsequent determinations when it is desirable to obtain a quantitative result.

Following the desired number of cycles, the reactions are stopped at 55°C by the addition of 8 ld of 95% formamide with 20 mM EDTA and 0.05% marker dyes per 10 tl reaction volume. Samples are not heated before loading onto denaturing polyacrylamide gels polyacrylamide, 19:1 crosslinking, 7 M urea, 89 mM tris-borate, pH 8.3, 2.8 mM EDTA). The samples were not heated to allow for the resolution of single-stranded and reduplexed uncleaved hairpin molecules.

The hairpin molecules may be attached to separate solid support molecules, such as agarose. styrene or magnetic beads, via the 3' end of each hairpin. A spacer molecule may be placed between the 3' end of the hairpin and the bead if so desired. The advantage of attaching the hairpins to a solid support is that this prevents the hybridization of the A- and T-hairpins to one another during the cycles of melting and annealing. The A- and T-hairpins are complementary to one another (as shown in Fig. 20D) and if allowed to anneal to one another over their entire lengths this would reduce the amount of hairpins available for hybridization to the alpha and tau primers during the detection reaction.

The 5' nucleases of the present invention are used in this assay because they lack significant synthetic activity. The lack of synthetic activity results in the production of a single cleaved hairpin product (as shown in Fig. 19B, lane Multiple cleavage products may be generated by 1) the presence of interfering synthetic activity (see Fig. 19B, lanes 6 and 8) or 2) the presence of primer-independent cleavage in the reaction. The presence of primer-independent cleavage is detected in the trigger/detection assay by the presence of different sized products at the fork of the cleavage structure. Primer-independent cleavage can be dampened or repressed, when present, by the use of uncleavable nucleotides in the fork o 25 region of the hairpin molecule. For example, thiolated nucleotides can be used to replace several nucleotides at the fork region to prevent primer-independent cleavage.

EXAMPLE Cleavage Of Linear Nucleic Acid Substrates From the above, it should be clear that native "wild type") thermostable DNA polymerases are capable of cleaving hairpin structures in a specific manner and that this discovery can be applied with success to a detection assay. In this example, the mutant S DNAPs of the present invention are tested against three different cleavage structures shown in 119- Fig. 22A. Structure 1 in Fig. 22A is simply single stranded 206-mer (the preparation and sequence information for which was discussed above). Structures 2 and 3 are duplexes: structure 2 is the same hairpin structure as shown in Fig. 12A (bottom), while structure 3 has the hairpin portion of Istr cnre 2 remov;d.

The cleavage reactions comprised 0.01 pmoles of the resulting substrate DNA, and 1 pmole of pilot oligonucleotide in a total volume of 10 pl of 10 mM Tris-Cl, pH 8.3, 100 mM KCI. 1 mM MgCl,. Reactions were incubated for 30 minutes at 55 0 C, and stopped by the addition of 8 pl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75 C for 2 minutes immediately before electrophoresis through a polyacrylamide gel (19:1 cross link), with 7M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

The results were visualized by autoradiography and are shown in Fig. 22B with the enzymes indicated as follows: I is native Taq DNAP; II is native Tfl DNAP; III is Cleavase® BX shown in Fig. 4E; IV is Cleavase® BB shown in Fig. 4F; V is the mutant shown in Fig. 5B; and VI is Cleavase® BN shown in Fig. 4G.

Structure 2 was used to "normalize" the comparison. For example, it was found that it took 50 ng of Taq DNAP and 300 ng of Cleavase® BN to give similar amounts of cleavage of Structure 2 in thirty (30) minutes. Under these conditions native Taq DNAP is unable to cleave Structure 3 to any significant degree. Native Tfl DNAP cleaves Structure 3 in a manner that creates multiple products.

By contrast, all of the mutants tested cleave the linear duplex of Structure 3. This finding indicates that this characteristic of the mutant DNA polymerases is consistent of thermostable polymerases across thermophilic species.

The finding described herein that the mutant DNA polymerases of the present 25 invention are capable of cleaving linear duplex structures allows for application to a more straightforward assay design (Fig. IA). Fig. 23 provides a more detailed schematic corresponding to the assay design of Fig. 1A.

''The two 43-mers depicted in Fig. 23 were synthesized by standard methods. Each included a fluorescein on the 5'end for detection purposes and a biotin on the 3' end to allow 30 attachment to streptavidin coated paramagnetic particles (the biotin-avidin attachment is indicated by the zig-zag line).

120 Before the trityl groups were removed, the oligonucleotides were purified by HPLC to remove truncated by-products of the synthesis reaction. Aliquots of each 43-mer were bound to M-280 Dynabeads (Dynal) at a density of 100 pmoles per mg of beads. Two mgs of bea-s (200 were wshed twice in iX wash/bind buffer (1 M NaCI, 5 mM Tris-Cl. pH 0.5 mM EDTA) with 0.1% BSA, 200 pl per wash. The beads were magnetically sedimented between washes to allow supernatant removal. After the second wash, the beads were resuspended in 200 pl of 2X wash/bind buffer (2 M Na Cl, 10 mM Tris-Cl, pH 7.5 with 1 mM EDTA), and divided into two 100 pl aliquots. Each aliquot received 1 pl of a 100 pM solution of one of the two oligonucleotides. After mixing, the beads were incubated at room temperature for 60 minutes with occasional gentle mixing. The beads were then sedimented and analysis of the supernatants showed only trace amounts of unbound oligonucleotide, indicating successful binding. Each aliquot of beads was washed three times, 100 pi per wash, with IX wash/bind buffer, then twice in a buffer of 10 mM Tris-Cl, pH 8.3 and 75 mM KCI. The beads were resuspended in a final volume of 100 pl of the Tris/KCI, for a concentration of 1 pmole of oligonucleotide bound to 10 jg of beads per pl of suspension.

The beads were stored at 4 0 C between uses.

The types of beads correspond to Fig. 1A. That is to say, type 2 beads contain the oligonucleotide (SEQ ID NO:33) comprising the complementary sequence (SEQ ID NO:34) for the alpha signal oligonucleotide (SEQ ID NO:35) as well as the beta signal oligonucleotide (SEQ ID NO:36) which when liberated is a 24-mer. This oligonucleotide has no "As" and is rich. Type 3 beads contain the oligonucleotide (SEQ ID NO:37) comprising the complementary sequence (SEQ ID NO:38) for the beta signal oligonucleotide (SEQ ID NO:39) as well as the alpha signal oligonucleotide (SEQ ID NO:35) which when liberated is a 20-mer. This oligonucleotide has no "Ts" and is rich.

*25 Cleavage reactions comprised 1 pl of the indicated beads, 10 pmoles of unlabelled alpha signal oligonucleotide as "pilot" (if indicated) and 500 ng of Cleavase® BN in 20 pl of 75 mM KC1, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCI 2 and 10 pM CTAB. All components except the enzyme were assembled, overlaid with light mineral oil and warmed to 53 0 C. The reactions were initiated by the addition of prewarmed enzyme and incubated at that 30 temperature for 30 minutes. Reactions were stopped at temperature by the addition of 16 pl of 95% formamide with 20 mM EDTA and 0.05% each of bromophenol blue and xylene cyanol. This addition stops the enzyme activity and, upon heating, disrupts the biotin-avidin i* link, releasing the majority (greater than 95%) of the oligonucleotides from the beads.

121 Samples were heated to 75°C for 2 minutes immediately before electrophoresis through a polyacrylamide gel (19:1 cross link), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Results were visualized by contact transfer of the resolved DNA to ositively cha.rged iiylun membrane and probing of the blocked membrane with an antifluorescein antibody conjugated to alkaline phosphatase. After washing, the signal was developed by incubating the membrane in Western Blue (Promega) which deposits a purple precipitate where the antibody is bound.

Fig. 24 shows the propagation of cleavage of the linear duplex nucleic acid structures of Fig. 23 by the DNAP mutants of the present invention. The two center lanes contain both types of beads. As noted above, the beta signal oligonucleotide (SEQ ID NO:36) when liberated is a 24-mer and the alpha signal oligonucleotide (SEQ ID NO:35) when liberated is a 20-mer. The formation of the two lower bands corresponding to the 24-mer and 20-mer is clearly dependent on "pilot".

EXAMPLE 6 Exonucleolytic Cleavage ("Nibbling") By Thermostable DNAPs It has been found that thermostable DNAPs, including those of the present invention.

have a true 5' exonuclease capable of nibbling the 5' end of a linear duplex nucleic acid structures. In this example, the 206 base pair DNA duplex substrate is again employed (see above). In this case, it was produced by the use of one 32 P-labeled primer and one unlabeled primer in a polymerase chain reaction. The cleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (with the unlabeled strand also present), 5 pmoles of pilot oligonucleotide (see pilot oligonucleotides in Fig. 12A) and 0.5 units of DNAPTaq or 0.5 p of Cleavase® BB in the E. coli extract (see above), in a total volume of 10 pi of 25 mM Tris-Cl, pH 8.5, 50 mM KCI, 1.5 mM MgCI,.

Reactions were initiated at 65 0 C by the addition of pre-warmed enzyme, then shifted to the final incubation temperature for 30 minutes. The results are shown in Fig. SoSamples in lanes 1-4 are the results with native Taq DNAP, while lanes 5-8 shown the results with Cleavase® BB. The reactions for lanes 1, 2, 5, and 6 were performed at 65 0 C and 30 reactions for lanes 3, 4, 7, and 8 were performed at 50 0 C and all were stopped at temperature o by the addition of 8 p1 of 95% formamide with 20 mM EDTA and 0.05% marker dyes.

Samples were heated to 75 0 C for 2 minutes immediately before electrophoresis through a Sacrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris*Borate, pH 8.3, 122 1.4 mM EDTA. The expected product in reactions 1, 2, 5, and 6 is 85 nucleotides long; in reactions 3 and 7, the expected product is 27 nucleotides long. Reactions 4 and 8 were performed without pilot, and should remain at 206 nucleotides. The faint band seen at 24 r.uclectides .is residual ed-labeied pinner from the PCR.

The surprising result is that Cleavase® BB under these conditions causes all of the label to appear in a very small species, suggesting the possibility that the enzyme completely hydrolyzed the substrate. To determine the composition of the fastest-migrating band seen in lanes 5-8 (reactions performed with the deletion mutant), samples of the 206 base pair duplex were treated with either T7 gene 6 exonuclease (USB) or with calf intestine alkaline phosphatase (Promega), according to manufacturers' instructions, to produce either labeled mononucleotide (lane a of Fig. 25B) or free 3 P-labeled inorganic phosphate (lane b of Fig. 25B), respectively. These products, along with the products seen in lane 7 of panel A were resolved by brief electrophoresis through a 20% acrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45 mM Tris*Borate, pH 8.3, 1.4 mM EDTA. Cleavase® BB is thus capable of converting the substrate to mononucleotides.

EXAMPLE 7 Nibbling Is Duplex Dependent The nibbling by Cleavase® BB is duplex dependent. In this example, internally labeled, single strands of the 206-mer were produced by 15 cycles of primer extension incorporating a-"P labeled dCTP combined with all four unlabeled dNTPs, using an unlabeled 206-bp fragment as a template. Single and double stranded products were resolved by electrophoresis through a non-denaturing 6% polyacrylamide gel (29:1 cross-link) in a buffer of 45 mM Tris*Borate, pH 8.3, 1.4 mM EDTA, visualized by autoradiography, excised 25 from the gel, eluted by passive diffusion, and concentrated by ethanol precipitation.

The cleavage reactions comprised 0.04 pmoles of substrate DNA, and 2 ll of Cleavase® BB (in an E. coli extract as described above) in a total volume of 40 Pl of 10 mM pH 8.5, 50 mM KCI, 1.5 mM MgCI 2 Reactions were initiated by the addition of pre-warmed enzyme; 10 pl aliquots were removed at 5, 10, 20, and 30 minutes, and 30 transferred to prepared tubes containing 8 tl of 95% formamide with 30 mM EDTA and c 0.05% marker dyes. Samples were heated to 75 0 C for 2 minutes immediately before electrophoresis through a 10% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris*Borate, pH 8.3, 1.4 mM EDTA. Results were visualized by autoradiography 123 as shown in Fig. 26. Clearly, the cleavage by Cleavase® BB depends on a duplex structure: no cleavage of the single strand structure is detected whereas cleavage of the 206-mer duplex is complete.

EXAMPLE 8 Nibbling Can Be Target Directed The nibbling activity of the DNAPs of the present invention can be employed with success in a detection assay. One embodiment of such an assay is shown in Fig. 27. In this assay, a labeled oligontcleotide is employed that is specific for a target sequence. The oligonucleotide is in excess of the target so that hybridization is rapid. In this embodiment, the oligonucleotide contains two fluorescein labels whose proximity on the oligonucleotide causes their emission to be quenched. When the DNAP is permitted to nibble the oligonucleotide the labels separate and are detectable. The shortened duplex is destabilized and disassociates. Importantly, the target is now free to react with an intact labeled oligonucleotide. The reaction can continue until the desired level of detection is achieved.

An analogous, although different, type of cycling assay has been described employing lambda exonuclease. See C.G. Copley and C. Boot, BioTechniques 13:888 (1992).

The success of such an assay depends on specificity. In other words, the oligonucleotide must hybridize to the specific target. It is also preferred that the assay be sensitive; the oligonucleotide ideally should be able to detect small amounts of target.

Fig. 28A shows a 5'-end 32 P-labelled primer bound to a plasmid target sequence. In this case.

the plasmid was pUC19 (commercially available) which was heat denatured by boiling two minutes and then quick chilling. The primer is a 21-mer (SEQ ID NO:39). The enzyme .employed was Cleavase® BX (a dilution equivalent to 5 x 10 3 p.l extract) in 100 mM KCI, 10 mM Tris-Cl, pH 8.3, 2 mM MnCI 2 The reaction was performed at 55 0 C for sixteen (16) hours with or without genomic background DNA (from chicken blood). The reaction was stopped by the addition of 8 pi of 95% formamide with 20 mM EDTA and marker dyes.

The products of the reaction were resolved by PAGE (10% polyacrylamide, 19:1 cross link, 1 x TBE) as seen in Fig. 28B. Lane contains the labeled 21-mer. Lanes 1-3 30 contain no specific target, although Lanes 2 and 3 contain 100 ng and 200 ng of genomic DNA, respectively. Lanes 4, 5 and 6 all contain specific target with either 0 ng, 100 ng or 200 ng of genomic DNA, respectively. It is clear that conversion to mononucleotides occurs S124 124 in Lanes 4, 5 and 6 regardless of the presence or amount of background DNA. Thus. the nibbling can be target directed and specific.

EXAIVMPLE 9 Cleavase® Purification As noted above, expressed thermostable proteins the 5' nucleases), were isolated by crude bacterial cell extracts. The precipitated E coli proteins were then, along with other cell debris, removed by centrifugation. In this example, cells expressing the BN clone were cultured and collected (500 grams). For each gram (wet weight) of E. coli, 3 ml of lysis buffer (50 mM Tris-HC1, pH 8.0, 1 mM EDTA, 100M NaCI) was added. The cells were lysed with 200 pg/ml lysozyme at room temperature for 20 minutes. Thereafter deoxycholic acid was added to make a 0.2% final concentration and the mixture was incubated 15 minutes at room temperature.

The lysate was sonicated for approximately 6-8 minutes at 0°C. The precipitate was removed by centrifugation (39,000g for 20 minutes). Polyethyleneimine was added to the supernatant and the mixture was incubated on ice for 15 minutes.

The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for 30 minutes at 60 0 C and then centrifuged again (5,000g for 15 minutes) and the supernatant was again retained.

The supernatant was precipitated with 35% ammonium sulfate at 4°C for 15 minutes.

The mixture was then centrifuged (5,000g for 15 minutes) and the supernatant was removed.

The precipitate was then dissolved in 0.25 M KCI, 20 Tris pH 7.6, 0.2% Tween and 0.1 EDTA) and then dialyzed against Binding Buffer (8X Binding Buffer comprises: imidazole, 4M NaCI, 160 mM Tris-HCl, pH 7.9).

25 The solubilized protein is then purified on the Ni- column (Novagen). The Binding Buffer is allows to drain to the top of the column bed and load the column with the prepared extract. A flow rate of about 10 column volumes per hour is optimal for efficient purification. If the flow rate is too fast, more impurities will contaminate the eluted fraction.

The column is washed with 25 ml (10 volumes) of IX Binding Buffer and then washed with 15 ml (6 volumes) of IX Wash Buffer (8X Wash Buffer comprises: 480mM imidazole. 4M NaCI, 160 mM Tris-HCI, pH The bound protein was eluted with 15 ml (6 volumes) of IX Elute Buffer (4X Elute Buffer comprises: 4 mM imidazole, 2 M NaCI, mM Tris-HC1, pH Protein is then reprecipitated with 35% Ammonium Sulfate as above.

*o° 125 The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100 mM KC1, ImM EDTA). The solution was brought up to 0.1% each of Tween 20 and NP-40 and stored at 4°C.

EXAMPLE The Use Of Various Divalent Cations In The Cleavage Reaction Influences The Nature Of The Resulting Cleavage Products In comparing the 5' nucleases generated by the modification and/or deletion of the Cterminal polymerization domain of Thermus aquaticus DNA polymerase (DNAPTaq), as diagrammed in Fig. 4B-G, significant differences in the strength of the interactions of these proteins with the 3' end of primers located upstream of the cleavage site (as depicted in Fig. 6) were noted. In describing the cleavage of these structures by Pol I-type DNA polymerases (Example 1 and Lyamichev et al., Science 260:778 [1993]). it was observed that in the absence of a primer, the location of the junction between the double-stranded region and the single-stranded 5' and 3' arms determined the site of cleavage, but in the presence of a primer, the location of the 3' end of the primer became the determining factor for the site of cleavage. It was postulated that this affinity for the 3' end was in accord with the synthesizing function of the DNA polymerase.

Structure 2, shown in Fig. 22A, was used to test the effects of a 3' end proximal to the cleavage site in cleavage reactions comprising several different solutions solutions containing different salts (KCI or NaCI), different divalent cations (Mn 2 or Mg 2 etc.] as well as the use of different temperatures for the cleavage reaction. When the reaction conditions were such that the binding of the enzyme a DNAP comprising a 5' nuclease, a modified DNAP or a 5' nuclease) to the 3' end (of the pilot oligonucleotide) near the 25 cleavage site was strong, the structure shown is cleaved at the site indicated in Fig. 22A.

This cleavage releases the unpaired 5' arm and leaves a nick between the remaining portion of the target nucleic acid and the folded 3' end of the pilot oligonucleotide. In contrast, when the reaction conditions are such that the binding of the DNAP (comprising a 5' nuclease) to the 3' end was weak, the initial cleavage was as described above, but after the release of the 30 5' arm, the remaining duplex is digested by the exonuclease function of the DNAP.

126 One way of weakening the binding of the DNAP to the 3' end is to remove all or part of the domain to which at least some of this function has been attributed. Some of nucleases created by deletion of the polymerization domain of DNAPTaq have enhanced true exonucleae function, as demonstrated in Ex. 6.

The affinity of these types of enzymes 5' nucleases associated with or derived from DNAPs) for recessed 3' ends may also be affected by the identity of the divalent cation present in the cleavage reaction. It was demonstrated by Longley et al. (Longley et al., Nucl.

Acids Res.. 18:7317 [1990]) that the use of MnCIl in a reaction with DNAPTaq enabled the polymerase to remove nucleotides from the 5' end of a primer annealed to a template, albeit inefficiently. Similarly, by examination of the cleavage products generated using Structure 2 from Fig. 22A, as described above, in a reaction containing either DNAPTaq or the Cleavase® BB nuclease, it was observed that the substitution of MnC1 2 for MgCI, in the cleavage reaction resulted in the exonucleolytic "nibbling" of the duplex downstream of the initial cleavage site. While not limiting the invention to any particular mechanism, it is thought that the substitution of MnCI, for MgCI 2 in the cleavage reaction lessens the affinity of these enzymes for recessed 3' ends.

In all cases, the use of MnCI enhances the 5' nuclease function, and in the case of the Cleavase® BB nuclease, a 50- to 100-fold stimulation of the 5' nuclease function is seen.

Thus, while the exonuclease activity of these enzymes was demonstrated above in the presence of MgCl 2 the assays described below show a comparable amount of exonuclease activity using 50 to 100-fold less enzyme when MnCI 2 is used in place of MgCI 2 When these reduced amounts of enzyme are used in a reaction mixture containing MgCIl, the nibbling or exonuclease activity is much less apparent than that seen in Examples 6-8.

Similar effects are observed in the performance of the nucleic acid detection assay 25 described in Examples 11-18 below when reactions performed in the presence of either MgCI, or MnCI, are compared. In the presence of either divalent cation, the presence of the invader oligonucleotide (described below) forces the site of cleavage into the probe duplex, but in the presence of MnCI 2 the probe duplex can be further nibbled producing a ladder of products that are visible when a 3' end label is present on the probe oligonucleotide. When the 30 invader oligonucleotide is omitted from a reaction containing Mn 2 the probe is nibbled from the 5' end. Mg 2 '-based reactions display minimal nibbling of the probe oligonucleotide. In any of these cases, the digestion of the probe is dependent upon the presence of the target nucleic acid. In the examples below, the ladder produced by the enhanced nibbling activity a a 127observed in the presence of Mn 2 is used as a positive indicator that the probe oligonucleotide has hybridized to the target sequence.

V%1 A X4flT I .V %Al L 1 1 i Invasive 5' Endonucleolytic Cleavage By Thermostable 5' Nucleases In The Absence of Polymerization As described in the examples above, 5' nucleases cleave near the junction between single-stranded and base-paired regions in a bifurcated duplex, usually about one base pair into the base-paired region. In this example, it is shown that thermostable 5' nucleases, including those of the present invention Cleavase® BN nuclease. Cleavase® A/G nuclease). have the ability to cleave a greater distance into the base paired region when provided with an upstream oligonucleotide bearing a 3' region that is homologous to a region of the subject duplex, as shown in Fig. Fig. 30 shows a synthetic oligonucleotide which was designed to fold upon itself which consists of the following sequence: TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3' (SEQ ID NO:40). This oligonucleotide is referred to as the "S-60 Hairpin." The 15 basepair hairpin formed by this oligonucleotide is further stabilized by a "tri-loop" sequence in the loop end three nucleotides form the loop portion of the hairpin) [Hiraro, I. el al. (1994) Nucleic Acids Res.

22(4):576]. Fig. 30 also show the sequence of the P-15 oligonucleotide and the location of the region of complementarity shared by the P-15 and S-60 hairpin oligonucleotides. The sequence of the P-15 oligonucleotide is 5'-CGAGAGACCACGCTG-3' (SEQ ID NO:41). As discussed in detail below, the solid black arrowheads shown in Fig. 29 indicate the sites of cleavage of the S-60 hairpin in the absence of the P-15 oligonucleotide and the hollow arrow S 25 heads indicate the sites of cleavage in the presence of the P-15 oligonucleotide. The size of the arrow head indicates the relative utilization of a particular site.

The S-60 hairpin molecule was labeled on its 5' end with biotin for subsequent detection. The S-60 hairpin was incubated in the presence of a thermostable 5' nuclease in the presence or the absence of the P-15 oligonucleotide. The presence of the full duplex which can be formed by the S-60 hairpin is demonstrated by cleavage with the Cleavase® BN 5' nuclease, in a primer-independent fashion in the absence of the P-15 oligonucleotide).

The release of 18 and 19-nucleotide fragments from the 5' end of the S-60 hairpin molecule 128 showed that the cleavage occurred near the junction between the single and double stranded regions when nothing is hybridized to the 3' arm of the S-60 hairpin (Fig. 31. lane 2).

The reactions shown in Fig. 31 were conducted as follows. Twenty fmole of the biotin-Iabeleu hairpin DNA (SEQ ID NO:40) was combined with 0.1 ng of Cleavase® BN enzyme and 1 pl of 100 mM MOPS (pH 7.5) containing 0.5% each of Tween-20 and in a total volume of 9 pl. In the reaction shown in lane 1, the enzyme was omitted and the volume was made up by addition of distilled water (this served as the uncut or no enzyme control). The reaction shown in lane 3 of Fig. 31 also included 0.5 pmole of the oligonucleotide (SEQ ID NO:41), which can hybridize to the unpaired 3' arm of the hairpin (SEQ ID NO:40), as diagrammed in Fig. The reactions were overlaid with a drop of mineral oil, heated to 95 0 C for 15 seconds, then cooled to 37 0 C, and the reaction was started by the addition of 1 il of 10 mM MnCI, to each tube. After 5 minutes, the reactions were stopped by the addition of 6 pl of formamide containing 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75 0

C

for 2 minutes immediately before electrophoresis through a 15% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

After electrophoresis, the gel plates were separated allowing the gel to remain flat on one plate. A 0.2 mm-pore positively-charged nylon membrane (NYTRAN, Schleicher and Schuell, Keene, NH), pre-wetted in H 2 0, was laid on top of the exposed gel. All air bubbles were removed. Two pieces of 3MM filter paper (Whatman) were then placed on top of the o. membrane, the other glass plate was replaced, and the sandwich was clamped with binder clips. Transfer was allowed to proceed overnight. After transfer, the membrane was carefully f peeled from the gel and allowed to air dry. After complete drying, the membrane was washed in 1.2X Sequenase Images Blocking Buffer (USB) using 0.3 ml of buffer/cm 2 of 25 membrane. The wash was performed for 30 minutes at room temperature. A streptavidin-alkaline phosphatase conjugate (SAAP, USB) was added to a 1:4000 dilution directly to the blocking solution, and agitated for 15 minutes. The membrane was rinsed briefly with H 2 0 and then washed three times for 5 minutes per wash using 0.5 ml/cm 2 of IX SAAP buffer (100 mM Tris-HCI, pH 10, 50 mM NaCI) with 0.1% sodium dodecyl sulfate 30 (SDS). The membrane was rinsed briefly with H 2 0 between each wash. The membrane was then washed once in IX SAAP buffer containing 1 mM MgCl2 without SDS, drained thoroughly and placed in a plastic heat-sealable bag. Using a sterile pipet, 5 mis of CDP-StarTM (Tropix, Bedford, MA) chemiluminescent substrate for alkaline phosphatase were 129added to the bag and distributed over the entire membrane for 2-3 minutes. The CDP-StarTM-treated membrane was exposed to XRP X-ray film (Kodak) for an initial exposure of 10 minutes.

Ihe resulting autoradiograph is shown in Fig. 31. In Fig. 31, the lane labeled "M" contains the biotinvlated P-15 oligonucleotide which served as a marker. The sizes (in nucleotides) of the uncleaved S-60 hairpin (60 nt; lane the marker (15 nt; lane and the cleavage products generated by cleavage of the S-60 hairpin in the presence (lane 3) or absence (lane 2) of the P-15 oligonucleotide are indicated.

Because the complementary regions of the S-60 hairpin are located on the same molecule, essentially no lag time should be needed to allow hybridization to form the duplex region of the hairpin). This hairpin structure would be expected to form long before the enzyme could locate and cleave the molecule. As expected, cleavage in the absence of the primer oligonucleotide was at or near the junction between the duplex and single-stranded regions, releasing the unpaired 5' arm (Fig. 31, lane The resulting cleavage products were 18 and 19 nucleotides in length.

It was expected that stability of the S-60 hairpin with the tri-loop would prevent the Poligonucleotide from promoting cleavage in the "primer-directed" manner described in Ex.

1 above, because the 3' end of the "primer" would remain unpaired. Surprisingly, it was found that the enzyme seemed to mediate an "invasion" by the P-15 primer into the duplex region of the S-60 hairpin, as evidenced by the shifting of the cleavage site 3 to 4 basepairs further into the duplex region, releasing the larger products (22 and 21 nt.) observed in lane 3 of Fig. 31.

The precise sites of cleavage of the S-60 hairpin are diagrammed on the structure in Fig. 30, with the solid black arrowheads indicating the sites of cleavage in the absence of the 25 P-15 oligonucleotide and the hollow arrow heads indicating the sites of cleavage in the presence of These data show that the presence on the 3' arm of an oligonucleotide having some sequence homology with the first several bases of the similarly oriented strand of the downstream duplex can be a dominant factor in determining the site of cleavage by 30 nucleases. Because the oligonucleotide which shares some sequence homology with the first several bases of the similarly oriented strand of the downstream duplex appears to invade the duplex region of the hairpin, it is referred to as an" invader" oligonucleotide. As shown in **the examples below, an invader oligonucleotide appears to invade (or displace) a region of** the examples below, an invader oligonucleotide appears to invade (or displace) a region of

S.

130 duplexed nucleic acid regardless of whether the duplex region is present on the same molecule a hairpin) or whether the duplex is formed between two separate nucleic acid strands.

EXAMPLE 12 The Invader Oligonucleotide Shifts The Site Of Cleavage In A Pre-Formed Probe/Target Duplex In Ex. 11 it was demonstrated that an invader oligonucleotide could shift the site at which a 5' nuclease cleaves a duplex region present on a hairpin molecule. In this example, the ability of an invader oligonucleotide to shift the site of cleavage within a duplex region formed between two separate strands of nucleic acid molecules was examined.

A single-stranded target DNA comprising the single-stranded circular M13mpl9 molecule and a labeled (fluorescein) probe oligonucleotide were mixed in the presence of the reaction buffer containing salt (KCI) and divalent cations (Mg 2 or Mn 2 to promote duplex formation. The probe oligonucleotide refers to a labeled oligonucleotide which is complementary to a region along the target molecule M13mpl9). A second oligonucleotide (unlabelled) was added to the reaction after the probe and target had been allowed to anneal. The second oligonucleotide binds to a region of the target which is located downstream of the region to which the probe oligonucleotide binds. This second oligonucleotide contains sequences which are complementary to a second region of the target 20 molecule. If the second oligonucleotide contains a region which is complementary to a portion of the sequences along the target to which the probe oligonucleotide also binds, this second oligonucleotide is referred to as an invader oligonucleotide (see Fig. 32c).

Fig. 32 depicts the annealing of two oligonucleotides to regions along the M13mpl9 target molecule (bottom strand in all three structures shown). In Fig. 32 only a 52 nucleotide portion of the M13mpl9 molecule is shown; this 52 nucleotide sequence is listed in SEQ ID NO:42. The probe oligonucleotide contains a fluorescein label at the 3' end; the sequence of the probe is 5'-AGAAAGGAAGGGAAGAAAGC GAAAGG-3' (SEQ ID NO:43). In Fig. 32, sequences comprising the second oligonucleotide, including the invader 'oligonucleotide are underlined. In Fig. 32a, the second oligonucleotide, which has the 30 sequence 5'-GACGGGGAAAGCCGGCGA ACG-3' (SEQ ID NO:44), is complementary to a different and downstream region of the target molecule than is the probe oligonucleotide S: (labeled with fluorescein or "Fluor"); there is a gap between the second, upstream oligonucleotide and the probe for the structure shown in Fig. 32a. In Fig. 32b, the second, 131 upstream oligonucleotide, which has the sequence 5'-GAAAGCCGGCGAACGTGGCG-3' (SEQ ID NO:45), is complementary to a different region of the target molecule than is the probe oligonucleotide. but in this case, the second oligonucleotide and the probe oligon.uclct dc abut one anioit~Lr (ihat is the 3' end of the second, upstream oligonucleotide is immediately adjacent to the 5' end of the probe such that no gap exists between these two oligonucleotides). In Fig. 32c, the second, upstream oligonucleotide (5'-GGCGAACGTGGCGAGAAAGGA-3' [SEQ ID NO:46]) and the probe oligonucleotide share a region of complementarity with the target molecule. Thus, the upstream oligonucleotide has a 3' arm which has a sequence identical to the first several bases of the downstream probe. In this situation, the upstream oligonucleotide is referred to as an "invader" oligonucleotide.

The effect of the presence of an invader oligonucleotide upon the pattern of cleavage in a probe/target duplex formed prior to the addition of the invader was examined. The invader oligonucleotide and the enzyme were added after the probe was allowed to anneal to the target and the position and extent of cleavage of the probe were examined to determine a) if the invader was able to shift the cleavage site to a specific internal region of the probe, and if the reaction could accumulate specific cleavage products over time, even in the absence of thermal cycling, polymerization, or exonuclease removal of the probe sequence.

The reactions were carried out as follows. Twenty pl each of two enzyme mixtures 20 were prepared, containing 2 4l of Cleavase® A/G nuclease extract (prepared as described in Ex. with or without 50 pmole of the invader oligonucleotide (SEQ ID NO:46), as indicated, per 4 il of the mixture. For each of the eight reactions shown in Fig. 33. 150 fmole of M13mpl9 single-stranded DNA (available from Life Technologies, Inc.) was combined with 5 pmoles of fluorescein labeled probe (SEQ ID NO:43), to create the structure shown in Fig. 31c, but without the invader oligonucleotide present (the probe/target mixture).

One half (4 tubes) of the probe/target mixtures were combined with 1 pl of 100 mM MOPS, pH 7.5 with 0.5% each of Tween-20 and NP-40, 0.5 .pl of 1 M KCI and 0.25 pi of 80 mM MnCI,, and distilled water to a volume of 6 1 The second set of probe/target mixtures were combined with 1 .tl of 100 mM MOPS, pH 7.5 with 0.5% each of Tween-20 and NP-40, pl of 1 M KCI and 0.25 p1 of 80 mM MgCI 2 The second set of mixtures therefore contained MgCI, in place of the MnCI, present in the first set of mixtures.

•0• 132 The mixtures (containing the probe/target with buffer, KCI and divalent cation) were covered with a drop of ChillOut® evaporation barrier (MJ Research) and were brought to 0 C for 5 minutes to allow annealing. Four pl of the above enzyme mixtures without the invader oligonucleotide was added to reactions whose products are shown in lanes 1, 3, 5 and 7 of Fig. 33. Reactions whose products are shown lanes 2, 4, 6, and 8 of Fig. 33 received the same amount of enzyme mixed with the invader oligonucleotide (SEQ ID NO:46). Reactions 1, 2, 5 and 6 were incubated for 5 minutes at 60 0 C and reactions 3, 4, 7 and 8 were incubated for 15 minutes at 60 0

C.

All reactions were stopped by the addition of 8 .l of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 90 0 C for 1 minute immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked), containing 7 M urea.

in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the reaction products and were visualized by the use of an Hitachi FMBIO fluorescence imager, the output of which is seen in Fig. 33. The very low molecular weight fluorescent material seen in all lanes at or near the salt front in Fig. 33 and other fluoro-imager figures is observed when fluorescently-labeled oligonucleotides are electrophoresed and imaged on a fluoroimager. This material is not a product of the cleavage reaction.

The use of MnCI 2 in these reactions (lanes 1-4) stimulates the true exonuclease or "nibbling" activity of the Cleavase® enzyme, as described in Ex. 7, as is clearly seen in lanes 20 1 and 3 of Fig. 33. This nibbling of the probe oligonucleotide (SEQ ID NO:43) in the absence of invader oligonucleotide (SEQ ID NO:46) confirms that the probe oligonucleotide is forming a duplex with the target sequence. The ladder-like products produced by this nibbling reaction may be difficult to differentiate from degradation of the probe by nucleases that might be present in a clinical specimen. In contrast, introduction of the invader oligonucleotide (SEQ ID NO:46) caused a distinctive shift in the cleavage of the probe, pushing the site of cleavage 6 to 7 bases into the probe, confirming the annealing of both oligonucleotides. In presence of MnCI 2 the exonuclease "nibbling" may occur after the invader-directed cleavage event, until the residual duplex is destabilized and falls apart.

In a magnesium based cleavage reaction (lanes the nibbling or true exonuclease 30 function of the Cleavase® A/G is enzyme suppressed (but the endonucleolytic function of the enzyme is essentially unaltered), so the probe oligonucleotide is not degraded in the absence of the invader (Fig. 33, lanes 5 and When the invader is added, it is clear that the invader oligonucleotide can promote a shift in the site of the endonucleolytic cleavage of the annealed 133 probe. Comparison of the products of the 5 and 15 minute reactions with invader (lanes 6 and 8 in Fig. 33) shows that additional probe hybridizes to the target and is cleaved. The calculated melting temperature (Tm) of the portion of probe that is not invaded .nuc!etide.s 26 of SE ID is 56"C, so the observed turnover (as evidenced by the accumulation of cleavage products with increasing reaction time) suggests that the full length of the probe molecule, with a calculated Tm of 76 0 C, is must be involved in the subsequent probe annealing events in this 60 0 C reaction.

EXAMPLE 13 The Overlap Of The 3' Invader Oligonucleotide Sequence With The 5' Region Of The Probe Causes A Shift In The Site Of Cleavage In Ex. 12, the ability of an invader oligonucleotide to cause a shift in the site of cleavage of a probe annealed to a target molecule was demonstrated. In this example, experiments were conducted to examine whether the presence of an oligonucleotide upstream from the probe was sufficient to cause a shift in the cleavage site(s) along the probe or whether the presence of nucleotides on the 3' end of the invader oligonucleotide which have the same sequence as the first several nucleotides at the 5' end of the probe oligonucleotide were required to promote the shift in cleavage.

To examine this point, the products of cleavage obtained from three different arrangements of target-specific oligonucleotides are compared. A diagram of these oligonucleotides and the way in which they hybridize to a test nucleic acid. M13mpl9, is shown in Fig. 32. In Fig. 32a, the 3' end of the upstream oligonucleotide (SEQ ID NO:45) is located upstream of the 5' end of the downstream "probe" oligonucleotide (SEQ ID NO:43) such that a region of the M13 target which is not paired to either oligonucleotide is present.

In Fig. 32b, the sequence of the upstream oligonucleotide (SEQ ID NO:45) is immediately upstream of the probe (SEQ ID NO:43), having neither a gap nor an overlap between the sequences. Fig. 32c diagrams the arrangement of the substrates used in the assay of the present invention, showing that the upstream "invader" oligonucleotide (SEQ ID NO:46) has the same sequence on a portion of its 3' region as that present in the 5' region of the 30 downstream probe (SEQ ID NO:43). That is to say, these regions will compete to hybridize to the same segment of the M13 target nucleic acid.

o• 134- In these experiments, four enzyme mixtures were prepared as follows (planning 5 pl per digest): Mixture 1 contained 2.25pl of Cleavase® A/G nuclease extract (prepared as described in Ex. 2) per 5 pl of mixture, in 20 mM MOPS, pH 7.5 with 0.1 each of Tween and NP-40, 4 mM MnCI, and 100 mM KC1. Mixture 2 contained 11.25 units of Taq DNA polymerase (Promega Corp., Madison, WI) per 5 l of mixture in 20 mM MOPS, pH with 0.1 each of Tween 20 and NP-40, 4 mM MnCI, and 100 mM KCI. Mixture 3 contained 2.25 tl of Cleavase® A/G nuclease extract per 5 pl of mixture in 20 mM Tris-HC1, pH 8.5, 4 mM MgCl, and 100 mM KC1. Mixture 4 contained 11.25 units of Taq DNA polymerase per 5 p of mixture in 20 mM Tris-HC1, pH 8.5, 4 mM MgC1, and 100 mM KC1.

For each reaction. 50 fmole of M13mpl9 single-stranded DNA (the target nucleic acid) was combined with 5 pmole of the probe oligonucleotide (SEQ ID NO:43 which contained a fluorescein label at the 3' end) and 50 pmole of one of the three upstream oligonucleotides diagrammed in Fig. 32 one of SEQ ID NOS:44-46), in a total volume of 5 pl of distilled water. The reactions were overlaid with a drop of ChillOutTM evaporation barrier (MJ Research) and warmed to 62 0 C. The cleavage reactions were started by the addition of 5 pl of an enzyme mixture to each tube, and the reactions were incubated at 62 0

C

for 30 min. The reactions shown in lanes 1-3 of Fig. 34 received Mixture 1; reactions 4-6 received Mixture 2; reactions 7-9 received Mixture 3 and reactions 10-12 received Mixture 4.

After 30 minutes at 62 0 C, the reactions were stopped by the addition of 8 Pl of 20 formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75 0 C for 2 minutes immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked).

with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

Following electrophoresis, the products of the reactions were visualized by the use of an Hitachi FMBIO fluorescence imager, the output of which is seen in Fig. 34. The reaction products shown in lanes 1, 4, 7 and 10 of Fig. 34 were from reactions which contained SEQ ID NO:44 as the upstream oligonucleotide (see Fig. 32a). The reaction products shown in lanes 2, 5, 8 and 11 of Fig. 34 were from reactions which contained SEQ ID NO:45 as the upstream oligonucleotide (see Fig. 32b). The reaction products shown in lanes 3, 6, 9 and 12 of Fig. 34 were from reactions which contained SEQ ID NO:46, the invader oligonucleotide, 30 as the upstream oligonucleotide (see Fig. 32c).

Examination of the Mn 2 based reactions using either Cleavase® A/G nuclease or DNAPTaq as the cleavage agent (lanes 1 through 3 and 4 through 6, respectively) shows that both enzymes have active exonuclease function in these buffer conditions. The use of a 3' 135 label on the probe oligonucleotide allows the products of the nibbling activity to remain labeled, and therefore visible in this assay. The ladders seen in lanes 1, 2, 4 and 5 confirm that the probe hybridize to the target DNA as intended. These lanes also show that the Incation of the non-in;vasivc oligoinuclcuiius have iinie effect on the products generated. The uniform ladder created by these digests would be difficult to distinguish from a ladder causes by a contaminating nuclease, as one might find in a clinical specimen. In contrast, the products displayed in lanes 3 and 6, where an invader oligonucleotide was provided to direct the cleavage, show a very distinctive shift, so that the primary cleavage product is smaller than those seen in the non-invasive cleavage. This product is then subject to further nibbling in these conditions, as indicated by the shorter products in these lanes. These invader-directed cleavage products would be easily distinguished from a background of non-specific degradation of the probe oligonucleotide.

When Mg 2 is used as the divalent cation the results are even more distinctive. In lanes 7, 8, 10 and 11 of Fig. 34, where the upstream oligonucleotides were not invasive, minimal nibbling is observed. The products in the DNAPTaq reactions show some accumulation of probe that has been shortened on the 5' end by one or two nucleotides consistent with previous examination of the action of this enzyme on nicked substrates (Longley et al., supra). When the upstream oligonucleotide is invasive, however, the appearance of the distinctively shifted probe band is seen. These data clearly indicated that it is the invasive 3' portion of the upstream oligonucleotide that is responsible for fixing the site of cleavage of the downstream probe.

Thus. the above results demonstrate that it is the presence of the free or initially non- -annealed nucleotides at the 3' end of the invader oligonucleotide which mediate the shift in the cleavage site, not just the presence of an oligonucleotide annealed upstream of the probe.

Nucleic acid detection assays which employ the use of an invader oligonucleotide are termed "invader-directed cleavage" assays.

136i EXAMPLE 14 Invader-Directed Cleavage Recognizes Single And Double Stranded Target Molecules In A Background Of Non-Target DNA Molecules F-or a nucleic acid detection method to be broadly useful, it must be able to detect a specific target in a sample that may contain large amounts of other DNA, bacterial or human chromosomal DNA. The ability of the invader directed cleavage assay to recognize and cleave either single- or double-stranded target molecules in the presence of large amounts of non-target DNA was examined. In these experiments a model target nucleic acid, M13, in either single or double stranded form (single-stranded M13mpl8 is available from Life Technologies, Inc. and double-stranded Ml3mpl9 is available from New England Biolabs), was combined with human genomic DNA (Novagen, Madison, WI) and then utilized in invader-directed cleavage reactions. Before the start of the cleavage reaction, the DNAs were heated to 95°C for 15 minutes to completely denature the samples, as is standard practice in assays, such as polymerase chain reaction or enzymatic DNA sequencing, which involve solution hybridization of oligonucleotides to double-stranded target molecules.

For each of the reactions shown in lanes 2-5 of Fig. 35, the target DNA (25 fmole of the ss DNA or 1 pmole of the ds DNA) was combined with 50 pmole of the invader oligonucleotide (SEQ ID NO:46); for the reaction shown in lane I the target DNA was omitted. Reactions 1, 3 and 5 also contained 470 ng of human genomic DNA. These 20 mixtures were brought to a volume of 10 pl with distilled water, overlaid with a drop of ChillOutTM evaporation barrier (MJ Research), and brought to 95°C for 15 minutes. After this incubation period, and still at 95 0 C, each tube received 10 pl of a mixture comprising 2.25 pl of Cleavase® A/G nuclease extract (prepared as described in Ex. 2) and 5 pmole of the probe oligonucleotide (SEQ ID NO:43), in 20 mM MOPS, pH 7.5 with 0.1 each of Tween and NP-40, 4 mM MnCI 2 and 100 mM KC1. The reactions were brought to 62 0 C for minutes and stopped by the addition of 12 pl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75°C for 2 minutes immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions were visualized 30 by the use of an Hitachi FMBIO fluorescence imager. The results are displayed in Fig. In Fig. 35, lane I contains the products of the reaction containing the probe (SEQ ID NO:43), the invader oligonucleotide (SEQ ID NO:46) and human genomic DNA.

Examination of lane 1 shows that the probe and invader oligonucleotides are specific for the 137 target sequence, and that the presence of genomic DNA does not cause any significant background cleavage.

In Fig. 35, lanes 2 and 3 contain reaction products from reactions containing the single stranded targCt DNA (MI3ipi), the probe (SEQ 10 NO:43) and the invader oligonucleotide (SEQ ID NO:46) in the absence or presence of human genomic DNA, respectively. Examination of lanes 2 and 3 demonstrate that the invader detection assay may be used to detect the presence of a specific sequence on a single-stranded target molecule in the presence or absence of a large excess of competitor DNA (human genomic DNA).

In Fig. 35, lanes 4 and 5 contain reaction products from reactions containing the double-stranded target DNA (M13mpl9), the probe (SEQ ID NO:43) and the invader oligonucleotide (SEQ ID NO:46) in the absence or presence of human genomic DNA, respectively. Examination of lanes 4 and 5 show that double stranded target molecules are eminently suitable for invader-directed detection reactions. The success of this reaction using a short duplexed molecule. M13mpl9, as the target in a background of a large excess of genomic DNA is especially noteworthy as it would be anticipated that the shorter and less complex M 13 DNA strands would be expected to find their complementary strand more easily than would the strands of the more complex human genomic DNA. If the M13 DNA reannealed before the probe and/or invader oligonucleotides could bind to the target sequences along the M13 DNA, the cleavage reaction would be prevented. In addition, because the denatured genomic DNA would potentially contain regions complementary to the probe and/or invader oligonucleotides it was possible that the presence of the genomic DNA would inhibit the reaction by binding these oligonucleotides thereby preventing their hybridization to the M13 target. The above results demonstrate that these theoretical concerns are not a problem under the reaction conditions employed above.

In addition to demonstrating that the invader detection assay may be used to detect sequences present in a double-stranded target, these data also show that the presence of a large amount of non-target DNA (470 ng/20 pl1 reaction) does not lessen the specificity of the cleavage. While this amount of DNA does show some impact on the rate of product accumulation, probably by binding a portion of the enzyme, the nature of the target sequence, 30 whether single- or double-stranded nucleic acid, does not limit the application of this assay.

138- EXAMPLE Signal Accumulation In The Invader-Directed Cleavage Assay As A Function Of Target Concentration To investigate whether the invader-directed cleavage assay could be used to indicate the amount of target nucleic acid in a sample, the following experiment was performed.

Cleavage reactions were assembled which contained an invader oligonucleotide (SEQ ID NO:46), a labeled probe (SEQ ID NO:43) and a target nucleic acid, Ml3mpl9. A series of reactions, which contained smaller and smaller amounts of the MI3 target DNA, was employed in order to examine whether the cleavage products would accumulate in a manner that reflected the amount of target DNA present in the reaction.

The reactions were conducted as follows. A master mix containing enzyme and buffer was assembled. Each 5 pl of the master mixture contained 25 ng of Cleavase® BN nuclease in 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4 mM MnCI, and 100 mM KC1. For each of the cleavage reactions shown in lanes 4-13 of Fig. 36, a DNA mixture was generated which contained 5 pmoles of the fluorescein-labeled probe oligonucleotide (SEQ ID NO:43), 50 pmoles of the invader oligonucleotide (SEQ ID NO:46) and 100, 50, 1, 0.5, 0.1, 0.05, 0.01 or 0.005 fmoles of single-stranded M13mpl9, respectively, for every pl of the DNA mixture. The DNA solutions were covered with a drop of ChillOut" evaporation barrier (MJ Research) and brought to 61°C. The cleavage reactions were started 20 by the addition of 5 pl of the enzyme mixture to each of tubes (final reaction volume was pl). After 30 minutes at 61 0 C, the reactions were terminated by the addition of 8 pl of formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 90 0 C for 1 minutes immediately before electrophoresis through a 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate (pH 1.4 mM EDTA. To provide reference standards), 1.0, 0.1 and 0.01 pmole aliquots of fluorescein-labeled probe oligonucleotide (SEQ ID NO:43) were diluted with the above formamide solution to a final volume of 18 pl. These reference markers were loaded into lanes 1-3, respectively of the gel. The products of the cleavage reactions (as well as the Sreference standards) were visualized following electrophoresis by the use of a Hitachi FMBIO fluorescence imager. The results are displayed in Fig. 36.

In Fig. 36, boxes appear around fluorescein-containing nucleic acid the cleaved and uncleaved probe molecules) and the amount of fluorescein contained within each box is indicated under the box. The background fluorescence of the gel (see box labeled 139 "background") was subtracted by the fluoro-imager to generate each value displayed under a box containing cleaved or uncleaved probe products (the boxes are numbered 1-14 at top left with a V followed by a number below the box). The lane marked contains fluoresceinated nlignnurl et;ide which serv.ed as markers.

The results shown in Fig. 36. demonstrate that the accumulation of cleaved probe molecules in a fixed-length incubation period reflects the amount of target DNA present in the reaction. The results also demonstrate that the cleaved probe products accumulate in excess of the cop), number of the target. This is clearly demonstrated by comparing the results shown in lane 3, in which 10 fmole (0.01 pmole) of uncut probe are displayed with the results shown in 5, where the products which accumulated in response to the presence of 10 fmole of target DNA are displayed. These results show that the reaction can cleave hundreds of probe oligonucleotide molecules for each target molecule present, dramatically amplifying the targetspecific signal generated in the invader-directed cleavage reaction.

EXAMPLE 16 Effect Of Saliva Extract On The Invader-Directed Cleavage Assay For a nucleic acid detection method to be useful in a medical a diagnostic) setting, it must not be inhibited by materials and contaminants likely to be found in a typical clinical specimen. To test the susceptibility of the invader-directed cleavage assay to various materials, including but not limited to nucleic acids, glycoproteins and carbohydrates, likely to be found in a clinical sample, a sample of human saliva was prepared in a manner consistent with practices in the clinical laboratory and the resulting saliva extract was added to the invader-directed cleavage assay. The effect of the saliva extract upon the inhibition of cleavage and upon the specificity of the cleavage reaction was examined.

One and one-half milliliters of human saliva were collected and extracted once with an equal volume of a mixture containing phenol:chloroform:isoamyl alcohol (25:24:1). The ,resulting mixture was centrifuged in a microcentrifuge to separate the aqueous and organic phases. The upper, aqueous phase was transferred to a fresh tube. One-tenth volumes of 3 M !NaOAc were added and the contents of the tube were mixed. Two volumes of 100% ethyl alcohol were added to the mixture and the sample was mixed and incubated at room temperature for 15 minutes to allow a precipitate to form. The sample was centrifuged in a S: microcentrifuge at 13,000 rpm for 5 minutes and the supernatant was removed and discarded.

A milky pellet was easily visible. The pellet was rinsed once with 70% ethanol, dried under 140vacuum and dissolved in 200 p. of 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the saliva extract). Each jil of the saliva extract was equivalent to 7.5 pl of saliva. Analysis of the saliva extract by scanning ultraviolet spectrophotometry showed a peak absorbance at about 260 nm and indicated the presence of approximately 45 ng of total nucleic acid per pi of extract.

The effect of the presence of saliva extract upon the following enzymes was examined: Cleavase® BN nuclease, Cleavase® A/G nuclease and three different lots of DNAPTaq: AmpliTaq* (Perkin Elmer; a recombinant form of DNAPTaq), AmpliTaq® LD (Perkin-Elmer; a recombinant DNAPTaq preparation containing very low levels of DNA) and Taq DNA polymerase (Fisher). For each enzyme tested, an enzyme/probe mixture was made comprising the chosen amount of enzyme with 5 pmole of the probe oligonucleotide (SEQ ID NO:43) in pl of 20 mM MOPS (pH 7.5) containing 0.1% each of Tween 20 and NP-40, 4 mM MnCI 2 100 mM KCl and 100 pg/ml BSA. The following amounts of enzyme were used: ng of Cleavase® BN prepared as described in Ex. 9; 2 plI of Cleavase® A/G nuclease extract prepared as described in Ex. 2; 2.25 pl (11.25 polymerase units) the following DNA polymerases: AmpliTaq® DNA polymerase (Perkin Elmer); AmpliTaq® DNA polymerase LD (low DNA; from Perkin Elmer); Taq DNA polymerase (Fisher Scientific).

For each of the reactions shown in Fig. 37, except for that shown in lane 1, the target DNA (50 fmoles of single-stranded M13mpl9 DNA) was combined with 50 pmole of the 20 invader oligonucleotide (SEQ ID NO:46) and 5 pmole of the probe oligonucleotide (SEQ ID NO:43); target DNA was omitted in reaction 1 (lane Reactions 1, 3, 5, 7, 9 and 11 included 1.5 pl of saliva extract. These mixtures were brought to a volume of 5 pl with distilled water, overlaid with a drop of ChillOut® evaporation barrier (MJ Research) and brought to 95°C for 10 minutes. The cleavage reactions were then started by the addition of 5 pl of the desired enzyme/probe mixture; reactions 1, 4 and 5 received Cleavase® A/G ~nuclease. Reactions 2 and 3 received Cleavase® BN; reactions 6 and 7 received AmpliTaq®; reactions 8 and 9 received AmpliTaq® LD; and reactions 10 and 11 received Taq DNA Polymerase from Fisher Scientific.

The reactions were incubated at 63 0 C for 30 minutes and were stopped by the addition 30 of 6 pi of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75 0 C for 2 minutes immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions were visualized by the use of an Hitachi FMBIO 141 fluorescence imager, and the results are displayed in Fig. 37.

A pairwise comparison of the lanes shown in Fig. 37 without and with the saliva extract, treated with each of the enzymes, shows that the saliva extract has different effects on each of the enzmes. While the Cicavase, BN nuciease and the AmpliTaq® are significantly inhibited from cleaving in these conditions, the Cleavase® A/G nuclease and AmpliTaq® LD display little difference in the yield of cleaved probe. The preparation of Taq DNA polymerase from Fisher Scientific shows an intermediate response, with a partial reduction in the yield of cleaved product. From the standpoint of polymerization, the three DNAPTaq variants should be equivalent; these should be the same protein with the same amount of synthetic activity. It is possible that the differences observed could be due to variations in the amount of nuclease activity present in each preparation caused by different handling during purification, or by different purification protocols. In any case, quality control assays designed to assess polymerization activity in commercial DNAP preparations would be unlikely to reveal variation in the amount of nuclease activity present. If preparations of DNAPTaq were screened for full 5' nuclease activity fthe 5' nuclease activity was specifically quantitated), it is likely that the preparations would display sensitivities (to saliva extract) more in line with that observed using Cleavase® A/G nuclease, from which DNAPTaq differs by a very few amino acids.

It is worthy of note that even in the slowed reactions of Cleavase® BN and the DNAPTaq variants there is no noticeable increase in non-specific cleavage of the probe oligonucleotide due to inappropriate hybridization or saliva-borne nucleases.

EXAMPLE 17 Comparison Of Additional 5' Nucleases In The Invader-Directed Cleavage Assay A number of eubacterial Type A DNA polymerases Pol I type DNA polymerases) have been shown to function as structure specific endonucleases (Ex. 1 and Lyamichev el al., supra). In this example, it was demonstrated that the enzymes of this class can also be made to catalyze the invader-directed cleavage of the present invention, albeit not 30 as efficiently as the Cleavase® enzymes.

Cleavase® BN nuclease and Cleavase® A/G nuclease were tested along side three different thermostable DNA polymerases: Thermus aquaticus DNA polymerase (Promega), **Thermus thermophilus and Thermus flavus DNA polymerases (Epicentre). The enzyme 142 mixtures used in the reactions shown in lanes 1-11 of Fig. 38 contained the following, each in a volume of 5 pl: Lane 1: 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and 4 mM MnCl,, 100 mM KCIL Lane 2: 25 ng of Cleavase® BN nuclease in the same solution described for lane 1; Lane 3: 2.25 pl of Cleavase® A/G nuclease extract (prepared as described in Ex. in the same solution described for lane 1; Lane 4: 2.25 Pl of Cleavase® A/G nuclease extract in 20 mM Tris-Cl, (pH 4 mM MgCI 2 and 100 mM KCI; Lane 11.25 polymerase units of Taq DNA polymerase in the same buffer described for lane 4; Lane 6: 11.25 polymerase units of Tih DNA polymerase in the same buffer described for lane 1; Lane 7: 11.25 polymerase units of Tth DNA polymerase in a 2X concentration of the buffer supplied by the manufacturer, supplemented with 4 mM MnCl 2 Lane 8: 11.25 polymerase units of Tth DNA polymerase in a 2X concentration of the buffer supplied by the manufacturer, supplemented with 4 mM MgCl; Lane 9: 2.25 polymerase units of Tfl DNA polymerase in the same buffer described for lane 1; Lane 10: 2.25 polymerase units of Tfl polymerase in a 2X concentration of the buffer supplied by the manufacturer, supplemented with 4 mM MnCI,; Lane 11: 2.25 polymerase units of Tfl DNA polymerase in a 2X concentration of the buffer supplied by the manufacturer, supplemented with 4 mM MgCl 2 Sufficient target DNA, probe and invader for all 11 reactions was combined into a master mix. This mix contained 550 fmoles of single-stranded Ml3mpl9 target DNA. 550 pmoles of the invader oligonucleotide (SEQ ID NO:46) and 55 pmoles of the probe oligonucleotide (SEQ ID NO:43), each as depicted in Fig. 32c, in 55 upl of distilled water.

Five pl of the DNA mixture was dispensed into each of 11 labeled tubes and overlaid with a drop of ChillOut® evaporation barrier (MJ Research). The reactions were brought to 63°C and cleavage was started by the addition of 5 pl of the appropriate enzyme mixture. The reaction mixtures were then incubated at 63 0 C temperature for 15 minutes. The reactions were stopped by the addition of 8 pl of 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 90 0 C for 1 minute immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris- Borate (pH 1.4 mM EDTA. Following electrophoresis, the products of the reactions were visualized by the use of an Hitachi FMBIO fluorescence imager, and the results are 30 displayed in Fig. 38. Examination of the results shown in Fig. 38 demonstrates that all of the nucleases tested have the ability to catalyze invader-directed cleavage in at least one of the buffer systems tested. Although not optimized here, these cleavage agents are suitable for use in the methods of the present invention.

143 EXAMPLE 18 The Invader-Directed Cleavage Assay Can Detect Single Base Differences In Target Nucleic Acid Sequences The abiity of the ivader-dircted cleavage assay to detect single base mismatch mutations was examined. Two target nucleic acid sequences containing Cleavase® enzymeresistant phosphorothioate backbones were chemically synthesized and purified by polyacrylamide gel electrophoresis. Targets comprising phosphorothioate backbones were used to prevent exonucleolytic nibbling of the target when duplexed with an oligonucleotide.

A target oligonucleotide, which provides a target sequence that is completely complementary to the invader oligonucleotide (SEQ ID NO:46) and the probe oligonucleotide (SEQ ID NO:43), contained the following sequence: 5'-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3' (SEQ ID NO:47). A second target sequence containing a single base change relative to SEQ ID NO:47 was synthesized: 5'-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCC ACGTTCGCCGGC-3 (SEQ ID NO:48; the single base change relative to SEQ ID NO:47 is shown using bold and underlined type). The consequent mismatch occurs within the region of the target as represented in Fig. 29.

To discriminate between two target sequences which differ by the presence of a single mismatch), invader-directed cleavage reactions were conducted using two different reaction temperatures (55 0 C and 60 0 Mixtures containing 200 fmoles of either SEQ ID NO:47 or SEQ ID NO:48, 3 pmoles of fluorescein-labeled probe oligonucleotide (SEQ ID NO:43), 7.7 pmoles of invader oligonucleotide (SEQ ID NO:46) and 2 l of Cleavase® A/G nuclease extract (prepared as described in Ex. 2) in 9 pl of 10 mM MOPS (pH 7.4) with 50 mM KCI were assembled, covered with a drop of ChillOut® evaporation barrier (MJ Research) and brought to the appropriate reaction temperature. The cleavage reactions were initiated by the addition of 1 pl of 20 mM MgCI 2 After 30 minutes at either 55 0 C or 60 0 C, 10 pl of formamide with 20 mM EDTA and 0.05% marker dyes was added to stop the reactions. The reaction mixtures where then heated to 90 0 C for one minute prior to loading 4 pl onto denaturing polyacrylamide gels. The resolved reaction products were visualized using a 30 Hitachi FMBIO fluorescence imager. The resulting image is shown in Fig. 39.

In Fig. 39, lanes 1 and 2 show the products from reactions conducted at 55 0 C; lanes 3 and 4 show the products from reactions conducted at 60 0 C. Lanes 1 and 3 contained products from reactions containing SEQ ID NO:47 (perfect match to probe) as the target. Lanes 2 and 144- 4 contained products from reactions containing SEQ ID NO:48 (single base mis-match with probe) as the target. The target that does not have a perfect hybridization match complete complementarity) with the probe will not bind as strongly, the Tm of that duplex "wi!c I l-o tia, l hdie Tm of the same region it perfectly matched. The results presented here show that reaction conditions can be varied to either accommodate the mis-match by lowering the temperature of the reaction) or to exclude the binding of the mis-matched sequence by raising the reaction temperature).

The results shown in Fig. 39 demonstrate that the specific cleavage event which occurs in invader-directed cleavage reactions can be eliminated by the presence of a single base mismatch between the probe oligonucleotide and the target sequence. Thus, reaction conditions can be chosen so as to exclude the hybridization of mis-matched invader-directed cleavage probes thereby diminishing or even eliminating the cleavage of the probe. In an extension of this assay system, multiple cleavage probes, each possessing a separate reporter molecule a unique label), could also be used in a single cleavage reaction, to simultaneously probe for two or more variants in the same target region. The products of such a reaction would allow not only the detection of mutations which exist within a target molecule, but would also allow a determination of the relative concentrations of each sequence mutant and wild type or multiple different mutants) present within samples containing a mixture of target sequences.

When provided in equal amounts, but in a vast excess at least a 100-fold molar excess; typically at least 1 pmole of each probe oligonucleotide would be used when the target sequence was present at about 10 fmoles or less) over the target and used in optimized conditions. As discussed above, any differences in the relative amounts of the target variants will not affect the kinetics of hybridization, so the amounts of cleavage of each probe will *reflect the relative amounts of each variant present in the reaction.

The results shown in the example clearly demonstrate that the invader-directed cleavage reaction can be used to detect single base difference between target nucleic acids.

EXAMPLE 19 ~The Invader-Directed Cleavage Reaction Is 30 Insensitive To Large Changes In Reaction Conditions The results shown above demonstrated that the invader-directed cleavage reaction can be used for the detection of target nucleic acid sequences and that this assay can be used to detect single base difference between target nucleic acids. These results demonstrated that 145 nucleases Cleavase® BN, Cleavase® A/G, DNAPTaq, DNAPTth, DNAPTfl) could be used in conjunction with a pair of overlapping oligonucleotides as an efficient way to recognize nucleic acid targets. In the experiments below it is demonstrated that invasive cleavage reaction is relatively insensitive t 1 large c ges in cundiiions thereby making the method suitable for practice in clinical laboratories.

The effects of varying the conditions of the cleavage reaction were examined for their effect(s) on the specificity of the invasive cleavage and the on the amount of signal accumulated in the course of the reaction. To compare variations in the cleavage reaction a "standard" invader cleavage reaction was first defined. In each instance, unless specifically stated to be otherwise, the indicated parameter of the reaction was varied, while the invariant aspects of a particular test were those of this standard reaction. The results of these tests are shown in Figs. 42-51.

a) The Standard Invader-Directed Cleavage Reaction The standard reaction was defined as comprising 1 fmole of M13mpl8 single-stranded target DNA (New England Biolabs), 5 pmoles of the labeled probe oligonucleotide (SEQ ID NO:49), 10 pmole of the upstream invader oligonucleotide (SEQ ID NO:50) and 2 units of Cleavase® A/G in 10 pl of 10 mM MOPS, pH 7.5 with 100 mM KCI, 4 mM MnCI 2 and 0.05% each Tween-20 and Nonidet-P40. For each reaction, the buffers, salts and enzyme were combined in a volume of 5 pl; the DNAs (target and two oligonucleotides) were combined in 5 il of dHO and overlaid with a drop of ChillOut® evaporation barrier (MJ Research). When multiple reactions were performed with the same reaction constituents, S. 'these formulations were expanded proportionally.

Unless otherwise stated, the sample tubes with the DNA mixtures were warmed to 61°C, and the reactions were started by the addition of 5 ul of the enzyme mixture. After minutes at this temperature, the reactions were stopped by the addition of 8 ul of formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to 75 0 C for 2 minutes immediately before electrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions were visualized by the use of an Hitachi FMBIO fluorescence imager. In each 30 case, the uncut probe material was visible as an intense black band or blob, usually in the top half of the panel, while the desired products of invader specific cleavage were visible as one or two narrower black bands, usually in the bottom half of the panel. Under some reaction conditions, particularly those with elevated salt concentrations, a secondary cleavage product 146is also visible (thus generating a doublet). Ladders of lighter gray bands generally indicate either exonuclease nibbling of the probe oligonucleotide or heat-induced, non-specific breakage of the probe.

Fig. 4i depicts the annealing of the probe and invader oligonucleotides to regions along the M13mpl8 target molecule (the bottom strand). In Fig. 41 only a 52 nucleotide portion of the M13mpl8 molecule is shown; this 52 nucleotide sequence is listed in SEQ ID NO:42 (this sequence is identical in both M13mpl8 and M13mpl9). The probe oligonucleotide (top strand) contains a Cy3 amidite label at the 5' end; the sequence of the probe is 5'-AGAAAGGAAGGGAAGAAAGCGAAA GGT-3' (SEQ ID NO:49. The bold type indicates the presence of a modified base (2'-O-CH 3 Cy3 amidite (Pharmacia) is a indodicarbocyanine dye amidite which can be incorporated at any position during the synthesis of oligonucleotides; Cy3 fluoresces in the yellow region (excitation and emission maximum of 554 and 568 nm, respectively). The invader oligonucleotide (middle strand) has the following sequence: 5'-GCCGGCGAACGTGGCGAGAAAGGA-3' (SEQ ID b) KCI Titration Fig. 42 shows the results of varying the KCl concentration in combination with the use of 2 mM MnCI,, in an otherwise standard reaction. The reactions were performed in duplicate for confirmation of observations; the reactions shown in lanes 1 and 2 contained no added KC1, lanes 3 and 4 contained KCI at 5 mM, lanes 5 and 6 contained 25 mM KCI, lanes .o 20 7 and 8 contained 50 mM KC1, lanes 9 and 10 contained 100 mM KCl and lanes 11 and 12 contained 200 mM KCI. These results show that the inclusion of KCI allows the generation of a specific cleavage product. While the strongest signal is observed at the 100 mM KCI concentration, the specificity of signal in the other reactions with KCI at or above 25 mM indicates that concentrations in the full range 25-200 mM) may be chosen if it is so desirable for any particular reaction conditions.

As shown in Fig. 42, the invader-directed cleavage reaction requires the presence of salt KCI) for effective cleavage to occur. In other reactions, it has been found that KCI can inhibit the activity of certain Cleavase® enzymes when present at concentrations above oo* about 25 mM (For example, in cleavage reactions using the S-60 oligonucleotide shown in 30 Fig. 30, in the absence of primer, the Cleavase® BN enzyme loses approximately 50% of its activity in 50 mM KC1). Therefore, the use of alternative salts in the invader-directed 0 cleavage reaction was examined. In these experiments, the potassium ion was replaced with 0 147 either Na' or Li or the chloride ion was replaced with glutamic acid. The replacement of KCI with alternative salts is described below in sections c-e.

c) NaCI Titration M;g A. o4.S rcsu.ts Gi ALiLg valiu..U onceiitUiUiis UI Na iN 111 placCe uof KCi (lanes 3-10) in combination with the use 2 mM MnCI,, in an otherwise standard reaction, in comparison to the effects seen with 100 mM KCl (lanes 1 and The reactions analyzed in lanes 3 and 4 contained NaCI at 75 mM, lanes 5 and 6 contained 100 mM, lanes 7 and 8 contained 150 mM and lanes 9 and 10 contained 200 mM. These results show that NaCI can be used as a replacement for KCI in the invader-directed cleavage reaction the presence of NaCI, like KCI. enhances product accumulation).

d) LiCI Titration Fig. 44 shows the results of using various concentrations of LiCI in place of KCI (lanes 3-14) in otherwise standard reactions, compared to the effects seen with 100 mM KCI (lanes 1 and The reactions analyzed in lanes 3 and 4 contained LiCI at 25 mM, lanes and 6 contained 50 mM, lanes 7 and 8 contained 75 mM, lanes 9 and 10 contained 100 mM, lanes 11 and 12 contained 150 mM and lanes 13 and 14 contained 200 mM. These results demonstrate that LiCI can be used as a suitable replacement for KCI in the invader-directed cleavage reaction the presence of LiCI, like KCI, enhances product accumulation).

e) KGlu Titration Fig. 45 shows the results of using a glutamate salt of potassium (KGlu) in place of the more commonly used chloride salt (KCI) in reactions performed over a range of temperatures.

KGlu has been shown to be a highly effective salt source for some enzymatic reactions, S* showing a broader range of concentrations which permit maximum enzymatic activity [Leirmo et al. (1987) Biochem. 26:2095]. The ability of KGlu to facilitate the annealing of the probe S 25 and invader oligonucleotides to the target nucleic acid was compared to that of LiC1. In these experiments, the reactions were run for 15 minutes, rather than the standard 20 minutes. The reaction analyzed in lane 1 contained 150 mM LiCI and was run at 65 0 C; the reactions analyzed in lanes 2-4 contained 200 mM, 300 mM and 400 mM KGlu, respectively and were run at 65 0 C. The reactions analyzed in lanes 5-8 repeated the array of salt concentrations 30 used in lanes 1-4, but were performed at 67 0 C; lanes 9-12 show the same array run at 69 0

C

"and lanes 13-16 show the same array run at 71 0 C. The results shown in Fig. 45 demonstrate that KGlu was very effective as a salt in the invasive cleavage reactions. In addition, these o* 148 data show that the range of allowable KGlu concentrations was much greater than that of LiCI, with full activity apparent even at 400 mM KGlu.

f) MnCI, And MgCI, Titration And Ability To Replace MnCI, With MgCi, In some instances it may be desirable to perform the invasive cleavage reaction in the presence of Mg either in addition to, or in place of Mn 2 as the necessary divalent cation required for activity of the enzyme employed. For example, some common methods of preparing DNA from bacterial cultures or tissues use MgCI, in solutions which are used to facilitate the collection of DNA by precipitation. In addition, elevated concentrations greater than 5 mM) of divalent cation can be used to facilitate hybridization of nucleic acids, in the same way that the monovalent salts were used above, thereby enhancing the invasive cleavage reaction. In this experiment, the tolerance of the invasive cleavage reaction was examined for 1) the substitution of MgCI 2 for MnCI 2 and for the ability to produce specific product in the presence of increasing concentrations of MgCI, and MnCI 2 Fig. 46 shows the results of either varying the concentration of MnCI, from 2 mM to 8 mM, replacing the MnCI 2 with MgCl 2 at 2 to 4 mM, or of using these components in combination in an otherwise standard reaction. The reactions analyzed in lanes I and 2 contained 2 mM each MnCI, and MgCl 2 lanes 3 and 4 contained 2 mM MnCI, only, lanes and 6 contained 3 mM MnCIl, lanes 7 and 8 contained 4 mM MnCI 2 lanes 9 and contained 8 mM MnCl,. The reactions analyzed in lanes 11 and 12 contained 2 mM MgC, and lanes 13 and 14 contained 4 mM MgCI 2 These results show that both MnCI, and MgCl1 can be used as the necessary divalent cation to enable the cleavage activity of the Cleavase® A/G enzyme in these reactions and that the invasive cleavage reaction can tolerate a broad range of concentrations of these components.

25 In addition to examining the effects of the salt environment on the rate of product accumulation in the invasive cleavage reaction, the use of reaction constituents shown to be effective in enhancing nucleic acid hybridization in either standard hybridization assays blot hybridization) or in ligation reactions was examined. These components may act as volume excluders, increasing the effective concentration of the nucleic acids of interest and thereby enhancing hybridization, or they may act as charge-shielding agents to minimize repulsion between the highly charged backbones of the nucleic acids strands. The results of these experiments are described in sections g and h below.

0 149 g) Effect Of CTAB Addition The polycationic detergent cetyltrietheylammonium bromide (CTAB) has been shown to dramatically enhance hybridization of nucleic acids [Pontius and Berg (1991) Proc. Natl.

Acad. Sci. USA 88:8237]. The data shown in Fig. 47 depicts the results of adding the detergent CTAB to invasive cleavage reactions in which 150 mM LiCl was used in place of the KCI in otherwise standard reactions. Lane I shows unreacted uncut) probe, and the reaction shown in lane 1 is the LiCl-modified standard reaction without CTAB. The reactions analyzed in lanes 3 and 4 contained 100 pM CTAB, lanes 5 and 6 contained 200 p.M CTAB, lanes 7 and 8 contained 400 UtM CTAB, lanes 9 and 10 contained 600 p.M CTAB, lanes 11 and 12 contained 800 pM CTAB and lanes 13 and 14 contained 1 mM CTAB. These results showed that the lower amounts of CTAB may have a very moderate enhancing effect under these reaction conditions, and the presence of CTAB in excess of about 500 gM was inhibitory to the accumulation of specific cleavage product.

h) Effect Of PEG Addition Fig. 48 shows the effect of adding polyethylene glycol (PEG) at various percentage concentrations to otherwise standard reactions. The effects of increasing the reaction temperature of the PEG-containing reactions was also examined. The reactions assayed in lanes I and 2 were the standard conditions without PEG, lanes 3 and 4 contained 4% PEG, lanes 5 and 6 contained 8% PEG and lanes 7 and 8 contained 12% PEG. Each of the aforementioned reactions was performed at 61 0 C. The reactions analyzed in lanes 9, 10, 11 and 12 were performed at 65 and contained 8% and 12% PEG, respectively.

These results show that at all percentages tested, and at both temperatures tested, the inclusion of PEG substantially eliminated the production of specific cleavage product.

In addition to the data presented above effect of CTAB and PEG addition), the 25 presence of IX Denhardts in the reaction mixture was found to have no adverse effect upon the cleavage reaction [50X Denhardt's contains per 500 ml: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g BSA]. In addition the presence of each component of Denhardt's was examined individually Ficoll alone, polyvinylpyrrolidone alone, BSA alone) for the effect upon the invader-directed cleavage reaction; no adverse effect was observed.

30 i) Effect Of The Addition Of Stabilizing Agents i Another approach to enhancing the output of the invasive cleavage reaction is to enhance the activity of the enzyme employed, either by increasing its stability in the reaction environment or by increasing its turnover rate. Without regard to the precise mechanism by 150 which various agents operate in the invasive cleavage reaction, a number of agents commonly used to stabilize enzymes during prolonged storage were tested for the ability to enhance the accumulation of specific cleavage product in the invasive cleavage reaction.

Fig. 49 shows het effects of adding glycerol at 15% and of adding the detergents Tween-20 and Nonidet-P40 at alone or in combination, in otherwise standard reactions.

The reaction analyzed in lane I was a standard reaction. The reaction analyzed in lane 2 contained 1.5% NP-40, lane 3 contained 1.5% Tween 20, lane 4 contained 15% glycerol.

The reaction analyzed in lane 5 contained both Tween-20 and NP-40 added at the above concentrations, lane 6 contained both glycerol and NP-40, lane 7 contained both glycerol and Tween-20, and lane 8 contained all three agents. The results shown in Fig. 49 demonstrate that under these conditions these adducts had little or no effect on the accumulation of specific cleavage product.

Fig. 50 shows the effects of adding gelatin to reactions in which the salt identity and concentration were varied from the standard reaction. In addition, all of these reactions were performed at 65°C, instead of 61°C. The reactions assayed in lanes 1-4 lacked added KC1, and included 0.02%, 0.05%, 0.1% or 0.2% gelatin, respectively. Lanes 5, 6, 7 and 8 contained the same titration of gelatin, respectively, and included 100 mM KCI. Lanes 9, 11 and 12, also had the same titration of gelatin, and additionally included 150 mM LiCI in place of KCI. Lanes 13 and 14 show reactions that did not include gelatin, but which contained either 100 mM KCI or 150 mM LiCI, respectively. The results shown in Fig. demonstrated that in the absence of salt the gelatin had a moderately enhancing effect on the oooo accumulation of specific cleavage product, but when either salt (KCI or LiCI) was added to reactions performed under these conditions, increasing amounts of gelatin reduced the product accumulation.

j) Effect Of Adding Large Amounts Of Non-Target Nucleic Acid In detecting specific nucleic acid sequences within samples, it is important to determine if the presence of additional genetic material non-target nucleic acids) will .30 have a negative effect on the specificity of the assay. In this experiment, the effect of including large amounts of non-target nucleic acid, either DNA or RNA, on the specificity of the invasive cleavage reaction was examined. The data was examined for either an alteration 151 in the expected site of cleavage, or for an increase in the nonspecific degradation of the probe oligonucleotide.

Fig. 51 shows the effects of adding non-target nucleic acid genomic DNA or tj an invasive cleavage reaction performed at 65'C, with 150 mM LiCI in place of the KCI in the standard reaction. The reactions assayed in lanes 1 and 2 contained 235 and 470 ng of genomic DNA, respectively. The reactions analyzed in lanes 3, 4, 5 and 6 contained 100 ng, 200 ng, 500 ng and 1 jpg of tRNA, respectively. Lane 7 represents a control reaction which contained no added nucleic acid beyond the amounts used in the standard reaction.

The results shown in Fig. 51 demonstrate that the inclusion of non-target nucleic acid in large amounts could visibly slow the accumulation of specific cleavage product (while not limiting the invention to any particular mechanism, it is thought that the additional nucleic acid competes for binding of the enzyme with the specific reaction components). In additional experiments it was found that the effect of adding large amounts of non-target nucleic acid can be compensated for by increasing the enzyme in the reaction. The data shown in Fig. 51 also demonstrate that a key feature of the invasive cleavage reaction, the specificity of the detection, was not compromised by the presence of large amounts of non-target nucleic acid.

In addition to the data presented above, invasive cleavage reactions were run with succinate buffer at pH 5.9 in place of the MOPS buffer used in the "standard" reaction; no adverse effects were observed.

The data shown in Figs. 42-51 and described above demonstrate that the invasive cleavage reaction can be performed using a wide variety of reaction conditions and is therefore suitable for practice in clinical laboratories.

EXAMPLE 25 Detection Of RNA Targets By Invader-Directed Cleavage In addition to the clinical need to detect specific DNA sequences for infectious and genetic diseases, there is a need for technologies that can quantitatively detect target nucleic acids that are composed of RNA. For example, a number of viral agents, such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) have RNA genomic material, the 30 quantitative detection of which can be used as a measure of viral load in a patient sample.

Such information can be of critical diagnostic or prognostic value.

Hepatitis C virus (HCV) infection is the predominant cause of post-transfusion non-A, h non-B (NANB) hepatitis around the world. In addition, HCV is the major etiologic agent of e 152 hepatocellular carcinoma (HCC) and chronic liver disease world wide. The genome of HCV is a small (9.4 kb) RNA molecule. In studies of transmission of HCV by blood transfusion it has been found the presence of HCV antibody, as measured in standard immunological tests, does not always correlate with the infectivity of the sample, while the presence of HC V RNA in a blood sample strongly correlates with infectivity. Conversely, serological tests may remain negative in immunosuppressed infected individuals, while HCV RNA may be easily detected Cuthbert (1994) Clin. Microbiol. Rev. 7:505].

The need for and the value of developing a probe-based assay for the detection the HCV RNA is clear. The polymerase chain reaction has been used to detect HCV in clinical samples, but the problems associated with carry-over contamination of samples has been a concern. Direct detection of the viral RNA without the need to perform either reverse transcription or amplification would allow the elimination of several of the points at which existing assays may fail.

The genome of the positive-stranded RNA hepatitis C virus comprises several regions including 5' and 3' noncoding regions 5' and 3' untranslated regions) and a polyprotein coding region which encodes the core protein two envelope glycoproteins (El and E2/NSI) and six nonstructural glycoproteins (NS2-NS5b). Molecular biological analysis of the HCV genome has showed that some regions of the genome are very highly conserved between isolates, while other regions are fairly rapidly changeable. The 5' noncoding region (NCR) is the most highly conserved region in the HCV. These analyses have allowed these viruses to be divided into six basic genotype groups, and then further classified into over a Sdozen sub-types [the nomenclature and division of HCV genotypes is evolving; see Altamirano et al., J Infect. Dis. 171:1034 (1995) for a recent classification scheme].

In order to develop a rapid and accurate method of detecting HCV present in infected individuals, the ability of the invader-directed cleavage reaction to detect HCV RNA was examined. Plasmids containing DNA derived from the conserved 5'-untranslated region of six different HCV RNA isolates were used to generate templates for in vitro transcription. The HCV sequences contained within these six plasmids represent genotypes 1 (four sub-types represented; la, Ib, Ic, and Alc), 2, and 3. The nomenclature of the HCV genotypes used 30 herein is that of Simmonds et al. [as described in Altamirano et at., supra]. The Alc subtype was used in the model detection reaction described below.

153 a) Generation Of Plasmids Containing HCV Sequences Six DNA fragments derived from HCV were generated by RT-PCR using RNA extracted from serum samples of blood donors; these PCR fragments were a gift of Dr. M.

Altamirnnn (T Tn;v-ritv nf ,;t;cI r Altam (University of British Columbia.... .cuver). Thse PCR. fragments represent HCV sequences derived from HCV genotypes la, lb, Ic, Alc, 2c and 3a.

The RNA extraction, reverse transcription and PCR were performed using standard techniques (Altamirano et al., supra). Briefly, RNA was extracted from 100 pl of serum using guanidine isothiocyanate, sodium lauryl sarkosate and phenol-chloroform [Inchauspe et al., Hepatology 14:595 (1991)]. Reverse transcription was performed according to the manufacturer's instructions using a GeneAmp rTth reverse transcriptase RNA PCR kit (Perkin-Elmer) in the presence of an external antisense primer, HCV342. The sequence of the HCV342 primer is 5'-GGTTTTTCTTTGAGG TTTAG-3' (SEQ ID NO:51). Following termination of the RT reaction, the sense primer HCV7 [5'-GCGACACTCCACCATAGAT-3' (SEQ ID NO:52)] and magnesium were added and a first PCR was performed. Aliquots of the first PCR products were used in a second (nested) PCR in the presence of primers HCV46 [5'-CTGTCTTCACGCAGAAAGC-3' (SEQ ID NO:53)] and HCV308 CTACGAGACCTC-3' (SEQ ID NO:54)]. The PCRs produced a 281 bp product which corresponds to a conserved 5' noncoding region (NCR) region of HCV between positions 284 and -4 of the HCV genome (Altamirano et al., supra).

The six 281 bp PCR fragments were used directly for cloning or they were subjected to an additional amplification step using a 50 pl PCR comprising approximately 100 fmoles of DNA, the HCV46 and HCV308 primers at 0.1 pM, 100 pM of all four dNTPs and units of Taq DNA polymerase in a buffer containing 10 mM Tris-HC1, pH 8.3, 50 mM KC1, mM MgCI 2 and 0.1% Tween 20. The PCRs were cycled 25 times at 96 0 C for 45 sec., S 25 55 0 C for 45 sec. and 72 0 C for 1 min. Two microliters of either the original DNA samples or the reamplified PCR products were used for cloning in the linear pT7Blue T-vector (Novagen, z. Madison, WI) according to manufacturer's protocol. After the PCR products were ligated to the pT7Blue T-vector, the ligation reaction mixture was used to transform competent JM109 cells (Promega). Clones containing the pT7Blue T-vector with an insert were selected by the 30 presence of colonies having a white color on LB plates containing 40 pg/ml X-Gal, 40 Pg/ml IPTG and 50 plg/ml ampicillin. Four colonies for each PCR sample were picked and grown overnight in 2 ml LB media containing 50 plg/ml carbenicillin. Plasmid DNA was isolated using the following alkaline miniprep protocol. Cells from 1.5 ml of the overnight culture 154 were collected by centrifugation for 2 min. in a microcentrifuge (14K rpm), the supernatant was discarded and the cell pellet was resuspended in 50 pl TE buffer with 10 lg/ml RNAse A (Pharmacia). One hundred microliters of a solution containing 0.2 N NaOH, 1% SDS was added and the ceiis were iysed for 2 min. The lysate was gently mixed with 100 pl of 1.32 M potassium acetate, pH 4.8, and the mixture was centrifuged for 4 min. in a microcentrifuge (14K rpm); the pellet comprising cell debris was discarded. Plasmid DNA was precipitated from the supernatant with 200 pl ethanol and pelleted by centrifugation a microceftrifuge (14K rpm). The DNA pellet was air dried for 15 min. and was then redissolved in 50 pl TE buffer (10 mM Tris-HCI, pH 7.8, 1 mM EDTA).

b) Reamplification Of HCV Clones To Add The Phage T7 Promoter For Subsequent In Vitro Transcription To ensure that the RNA product of transcription had a discrete 3' end it was necessary to create linear transcription templates which stopped at the end of the HCV sequence. These fragments were conveniently produced using the PCR to reamplify the segment of the plasmid containing the phage promoter sequence and the HCV insert. For these studies, the clone of HCV type Alc was reamplified using a primer that hybridizes to the T7 promoter sequence: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO:55; "the T7 promoter primer") (Novagen) in combination with the 3' terminal HCV-specific primer HCV308 (SEQ ID NO:54). For these reactions, 1 p. of plasmid DNA (approximately 10 to 100 ng) was reamplified in a 200 pl PCR using the T7 and HCV308 primers as described above with the exception that 30 cycles of amplification were employed. The resulting amplicon was 354 bp in length. After amplification the PCR mixture was transferred to a fresh 1.5 ml microcentrifuge tube, the mixture was brought to a final concentration of 2 M NH4OAc, and the products were precipitated by the addition of one volume of 100% isopropanol.

25 Following a 10 min. incubation at room temperature, the precipitates were collected by centrifugation, washed once with 80% ethanol and dried under vacuum. The collected material was dissolved in 100 p1 nuclease-free distilled water (Promega).

Segments of RNA were produced from this amplicon by in vitro transcription using the RiboMAXTM Large Scale RNA Production System (Promega) in accordance with the 30 manufacturer's instructions, using 5.3 pg of the amplicon described above in a 100 p.1 reaction. The transcription reaction was incubated for 3.75 hours, after which the DNA template was destroyed by the addition of 5-6 pl of RQ1 RNAse-free DNAse (1 unit/pl) according to the RiboMAXTM kit instructions. The reaction was extracted twice with 155 phenol/chloroform/isoamyl alcohol (50:48:2) and the aqueous phase was transferred to a fresh microcentrifuge tube. The RNA was then collected by the addition of 10 pl of 3M NH,OAc, pH 5.2 and 110 pl of 100% isopropanol. Following a 5 min. incubation at 4°C, the precipitate was collected hy crntrifilgation, washed once .wih 80% t and dried

J

i er vacuum. The sequence of the resulting RNA transcript (HCVI.1 transcript) is listed in SEQ ID NO:56.

c) Detection Of The HCV1.1 Transcript In The Invader- Directed Cleavage Assay Detection of the HCVl.1 transcript was tested in the invader-directed cleavage assay using an HCV-specific probe oligonucleotide (5'-CCGGTCGTCCTGGCAAT XCC-3' [SEQ ID NO:57]); X indicates the presence of a fluorescein dye on an abasic linker) and an HCVspecific invader oligonucleotide (5'-GTTTATCCAAGAAAGGAC CCGGTC-3' [SEQ ID NO:58]) that causes a 6-nucleotide invasive cleavage of the probe.

Each 10 p1 of reaction mixture comprised 5 pmole of the probe oligonucleotide (SEQ ID NO:57) and 10 pmole of the invader oligonucleotide (SEQ ID NO:58) in a buffer of mM MOPS, pH 7.5 with 50 mM KCI, 4 mM MnCI 2 0.05% each Tween-20 and and 7.8 units RNasin® ribonuclease inhibitor (Promega). The cleavage agents employed were Cleavase® A/G (used at 5.3 ng/10 l1 reaction) or DNAPTth (used at 5 polymerase units/10 pl reaction). The amount of RNA target was varied as indicated below. When RNAse treatment is indicated, the target RNAs were pre-treated with 10 plg of RNase A (Sigma) at 37°C for min. to demonstrate that the detection was specific for the RNA in the reaction and not due to the presence of any residual DNA template from the transcription reaction. RNase-treated Saliquots of the HCV RNA were used directly without intervening purification.

For each reaction, the target RNAs were suspended in the reaction solutions as described above, but lacking the cleavage agent and the MnCI, for a final volume of 10 pl, with the invader and probe at the concentrations listed above. The reactions were warmed to 46 0 C and the reactions were started by the addition of a mixture of the appropriate enzyme with MnCl 2 After incubation for 30 min. at 46 0 C, the reactions were stopped by the addition of 8 pl of 95% formamide, 10 mM EDTA and 0.02% methyl violet (methyl violet loading buffer). Samples were then resolved by electrophoresis through a 15% denaturing 30 polyacrylamide gel (19:1 cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeled reaction products were visualized using the FMBIO-100 Image Analyzer (Hitachi), with the resulting imager scan shown in Fig. 52.

156 In Fig. 52, the samples analyzed in lanes 1-4 contained 1 pmole of the RNA target, the reactions shown in lanes 5-8 contained 100 fmoles of the RNA target and the reactions shown in lanes 9-12 contained 10 fmoles of the RNA target. All odd-numbered lanes depict reactivio perfornudc using Cleavascw AiG enzyme and aii even-numbered lanes depict reactions performed using DNAPTth. The reactions analyzed in lanes 1, 2, 5, 6, 9 and contained RNA that had been pre-digested with RNase A. These data demonstrate that the invasive cleavage reaction efficiently detects RNA targets and further, the absence of any specific cleavage signal in the RNase-treated samples confirms that the specific cleavage product seen in the other lanes is dependent upon the presence of input RNA.

EXAMPLE 21 The Fate Of The Target RNA In The Invader-Directed Cleavage Reaction In this example, the fate of the RNA target in the invader-directed cleavage reaction was examined. As shown above in Ex. ID, when RNAs are hybridized to DNA oligonucleotides, the 5' nucleases associated with DNA polymerases can be used to cleave the RNAs; such cleavage can be suppressed when the 5' arm is long or when it is highly structured [Lyamichev et al. (1993) Science 260:778 and U.S. Patent No. 5,422,253, the disclosure of which is herein incorporated by reference]. In this experiment, the extent to which the RNA target would be cleaved by the cleavage agents when hybridized to the detection oligonucleotides the probe and invader oligonucleotides) was examined using reactions similar to those described in Ex. 20, performed using fluorescein-labeled RNA as a target.

Transcription reactions were performed as described in Ex. 20 with the exception that 25 2% of the UTP in the reaction was replaced with fluorescein-12-UTP (Boehringer Mannheim) and 5.3 pg of the amplicon was used in a 100 pl reaction. The transcription reaction was incubated for 2.5 hours, after which the DNA template was destroyed by the addition of 5-6 pl of RQ1 RNAse-free DNAse (1 unit/gl) according to the RiboMAXO kit instructions. The organic extraction was omitted and the RNA was collected by the addition of 10 gl of 3M 30 NaOAc, pH 5.2 and 110 il of 100% isopropanol. Following a 5 min. incubation at 4 0 C, the precipitate was collected by centrifugation, washed once with 80% ethanol and dried under vacuum. The resulting RNA was dissolved in 100 pl of nuclease-free water. 50% of the sample was purified by electrophoresis through a 8% denaturing polyacrylamide gel (19:1 157 cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. The gel slice containing the full-length material was excised and the RNA was eluted by soaking the slice overnight at 4 0 C in 200 pl of 10 mM Tris-C1, pH 8.0, 0.1 mM EDTA .nd 0 M NaAc. Th.e P. A then preciptat-e by the additiun of 2.5 volumes of 100%1 ethanol. After incubation at -20 0 C for 30 min., the precipitates were recovered by centrifugation, washed once with 80% ethanol and dried under vacuum. The RNA was dissolved in 25 pl of nuclease-free water and then quantitated by UV absorbance at 260 nm.

Samples of the purified RNA target were incubated for 5 or 30 min. in reactions that duplicated the Cleavase® A/G and DNAPTth invader reactions described in Ex. 20 with the exception that the reactions lacked probe and invader oligonucleotides. Subsequent analysis of the products showed that the RNA was very stable, with a very slight background of nonspecific degradation, appearing as a gray background in the gel lane. The background was not dependent on the presence of enzyme in the reaction.

Invader detection reactions using the purified RNA target were performed using the probe/invader pair described in Ex. 20 (SEQ ID NOS:57 and 58). Each reaction included 500 fmole of the target RNA, 5 pmoles of the fluorescein-labeled probe and 10 pmoles of the invader oligonucleotide in a buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCI, 4 mM MnCI 2 0.05% each Tween-20 and Nonidet-P40 and 39 units RNAsin® (Promega). These components were combined and warmed to 50 0 C and the reactions were started by the addition of either 53 ng of Cleavase® A/G or 5 polymerase units of DNAPTth. The final reaction volume was 10 pl. After 5 min at 50 0 C, 5 il aliquots of each reaction were removed to tubes containing 4 pl of 95% formamide, 10 mM EDTA and 0.02% methyl violet. The remaining aliquot received a drop of ChillOut® evaporation barrier and was incubated for an additional 25 min. These reactions were then stopped by the addition of 4 pl 25 of the above formamide solution. The products of these reactions were resolved by electrophoresis through separate 20% denaturing polyacrylamide gels (19:1 cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeled reaction products were visualized using the FMBIO-100 Image Analyzer (Hitachi), with the resulting imager scans shown in Figs. 53A (5 min reactions) and 30 53B (30 min. reactions).

In Fig. 53 the target RNA is seen very near the top of each lane, while the labeled probe and its cleavage products are seen just below the middle of each panel. The FMBIO- 100 Image Analyzer was used to quantitate the fluorescence signal in the probe bands. In 158 each panel, lane 1 contains products from reactions performed in the absence of a cleavage agent, lane 2 contains products from reactions performed using Cleavase® A/G and lane 3 contains products from reactions performed using DNAPTth.

Quaniitaiion of ite fluorescence signal in the probe bands revealed that after a 5 min.

incubation, 12% or 300 fmole of the probe was cleaved by the Cleavase® A/G and 29% or 700 fmole was cleaved by the DNAPTth. After a 30 min. incubation, Cleavase® A/G had cleaved 32% of the probe molecules and DNAPTth had cleaved 70% of the probe molecules.

(The images shown in Figs. 53A and 53B were printed with the intensity adjusted to show the small amount of background from the RNA degradation, so the bands containing strong signals are saturated and therefore these images do not accurately reflect the differences in measured fluorescence) The data shown in Fig. 53 clearly shows that, under invasive cleavage conditions, RNA molecules are sufficiently stable to be detected as a target and that each RNA molecule can support many rounds of probe cleavage.

EXAMPLE 22 Titration Of Target RNA In The Invader-Directed Cleavage Assay One of the primary benefits of the invader-directed cleavage assay as a means for detection of the presence of specific target nucleic acids is the correlation between the amount of cleavage product generated in a set amount of time and the quantity of the nucleic acid of interest present in the reaction. The benefits of quantitative detection of RNA sequences was discussed in Ex. 20. In this example, we demonstrate the quantitative nature of the detection assay through the use of various amounts of target starting material. In addition to demonstrating the correlation between the amounts of input target and output cleavage product, these data graphically show the degree to which the RNA target can be recycled in this assay **The RNA target used in these reactions was the fluorescein-labeled material described in Ex. 21 SEQ ID NO:56). Because the efficiency of incorporation of the fluorescein- 30 12-UTP by the T7 RNA polymerase was not known, the concentration of the RNA was determined by measurement of absorbance at 260 nm, not by fluorescence intensity. Each reaction comprised 5 pmoles of the fluorescein-labeled probe (SEQ ID NO:57) and 10 pmoles of the invader oligonucleotide (SEQ ID NO:58) in a buffer of 10 mM MOPS, pH 7.5 with 159- 150 mM LiCI, 4 mM MnC1 2 0.05% each Tween-20 and Nonidet-P40 and 39 units of RNAsin® (Promega). The amount of target RNA was varied from 1 to 100 fmoles, as indicated below. These components were combined, overlaid with ChillOut" evaporation barrier (Mi Research) and warmed to 50"C; the reactions were started by the addition of either 53 ng of Cleavase® A/G or 5 polymerase units of DNAPTth, to a final reaction volume of 10 tl. After 30 minutes at 50 0 C, reactions were stopped by the addition of 8 pl of formamide, 10 mM EDTA and 0.02% methyl violet. The unreacted markers in lanes 1 and 2 were diluted in the same total volume (18 ul). The samples were heated to 90 0 C for 1 minute and 2.5 il of each of these reactions were resolved by electrophoresis through a denaturing polyacrylamide gel (19:1 cross link) with 7M urea in a buffer of 45 mM Tris- Borate, pH 8.3, 1.4 mM EDTA, and the labeled reaction products were visualized using the FMBIO-100 Image Analyzer (Hitachi), with the resulting imager scans shown in Fig. 54.

In Fig. 54, lanes 1 and 2 show 5 pmoles of uncut probe and 500 fmoles of untreated RNA, respectively. The probe is the very dark signal near the middle of the panel, while the RNA is the thin line near the top of the panel. These RNAs were transcribed with a 2% substitution of fluorescein-12-UTP for natural UTP in the transcription reaction. The resulting transcript contains 74 U residues, which would give an average of 1.5 fluorescein labels per molecule. With one tenth the molar amount of RNA loaded in lane 2, the signal in lane 2 should be approximately one seventh (0.15X) the fluorescence intensity of the probe in lane 1. Measurements indicated that the intensity was closer to one fortieth, indicating an efficiency of label incorporation of approximately 17%. Because the RNA concentration was verified by A260 measurement this does not alter the experimental observations below, but it should be noted that the signal from the RNA and the probes does not accurately reflect the relative amounts in the reactions.

25 The reactions analyzed in lanes 3 through 7 contained 1, 5, 10, 50 and 100 fmoles of target, respectively, with cleavage of the probe accomplished by Cleavase® A/G. The reactions analyzed in lanes 8 through 12 repeated the same array of target amounts, with cleavage of the probe accomplished by DNAPTth. The boxes seen surrounding the product bands show the area of the scan in which the fluorescence was measured for each reaction.

The number of fluorescence units detected within each box is indicated below each box; background florescence was also measured.

160- It can be seen by comparing the detected fluorescence in each lane that the amount of product formed in these 30 minute reactions can be correlated to the amount of target material. The accumulation of product under these conditions is slightly enhanced when DNAI'*lth is used as the cleavage agent, but the correlation with the amount of target present remains. This demonstrates that the invader assay can be used as a means of measuring the amount of target RNA within a sample.

Comparison of the fluorescence intensity of the input RNA with that of the'cleaved product shows that the invader-directed cleavage assay creates signal in excess of the amount of target, so that the signal visible as cleaved probe is far more intense than that representing the target RNA. This further confirms the results described in Ex. 20, in which it was demonstrated that each RNA molecule could be used many times.

EXAMPLE 23 Detection Of DNA By Charge Reversal The detection of specific targets is achieved in the invader-directed cleavage assay by the cleavage of the probe oligonucleotide. In addition to the methods described in the preceding examples, the cleaved probe may be separated from the uncleaved probe using the charge reversal technique described below. This novel separation technique is related to the observation that positively charged adducts can affect the electrophoretic behavior of small oligonucleotides because the charge of the adduct is significant relative to charge of the whole complex. Observations of aberrant mobility due to charged adducts have been reported in the literature, but in all cases found, the applications pursued by other scientists have involved making oligonucleotides larger by enzymatic extension. As the negatively charged nucleotides are added on, the positive influence of the adduct is reduced to insignificance. As a result, the effects of positively charged adducts have been dismissed and have received infinitesimal notice in the existing literature.

This observed effect is of particular utility in assays based on the cleavage of DNA molecules. When an oligonucleotide is shortened through the action of a Cleavase® enzyme or other cleavage agent, the positive charge can be made to not only significantly reduce the net negative charge, but to actually override it, effectively "flipping" the net charge of the labeled entity. This reversal of charge allows the products of target-specific cleavage to be partitioned from uncleaved probe by extremely simple means. For example, the products of cleavage can be made to migrate towards a negative electrode placed at any point in a 161 reaction vessel, for focused detection without gel-based electrophoresis. When a slab gel is used, sample wells can be positioned in the center of the gel, so that the cleaved and uncleaved probes can be observed to migrate in opposite directions. Alternatively, a traditional vertical gel cani be used, but with the electrodes reversed reiative to usual DNA gels the positive electrode at the top and the negative electrode at the bottom) so that the cleaved molecules enter the gel, while the uncleaved disperse into the upper reservoir of electrophoresis buffer.

An additional benefit of this type of readout is that the absolute nature of the partition of products from substrates means that an abundance of uncleaved probe can be supplied to drive the hybridization step of the probe-based assay, yet the unconsumed probe can be subtracted from the result to reduce background.

Through the use of multiple positively charged adducts, synthetic molecules can be constructed with sufficient modification that the normally negatively charged strand is made nearly neutral. When so constructed, the presence or absence of a single phosphate group can mean the difference between a net negative or a net positive charge. This observation has particular utility when one objective is to discriminate between enzymatically generated fragments of DNA, which lack a 3' phosphate, and the products of thermal degradation, which retain a 3' phosphate (and thus two additional negative charges).

a) Characterization Of The Products Of Thermal Breakage Of DNA Oligonucleotides Thermal degradation of DNA probes results in high background which can obscure signals generated by specific enzymatic cleavage, decreasing the signal-to-noise ratio. To better understand the nature of DNA thermal degradation products, we incubated the tetrachlorofluorescein (TET)-labeled oligonucleotides 78 (SEQ ID NO:59) and 79 (SEQ ID 25 NO:60) (100 pmole each) in 50 pl 10 mM NaCO 3 (pH 10.6), 50 mM NaCI at 90 0 C for 4 hours. To prevent evaporation of the samples, the reaction mixture was overlaid with 50 pl of ChillOut® 14 liquid wax (MJ Research). The reactions were then divided in two equal aliquots (A and Aliquot A was mixed with 25 pl of methyl violet loading buffer and Aliquot B was dephosphorylated by addition of 2.5 pl of 100 mM MgCI, and 1 pl of I unit/pl Calf Intestinal Alkaline Phosphatase (CIAP) (Promega), with incubation at 37 0 C for min. after which 25 tl of methyl violet loading buffer was added. One microliter of each sample was resolved by electrophoresis through a 12% polyacrylamide denaturing gel and imaged as described in Ex. 21; a 585 nm filter was used with the FMBIO Image Analyzer.

The resulting imager scan is shown in Fig. 55. In Fig. 55, lanes 1-3 contain the TET-labeled -162 t oligonucleotide 78 and lanes 4-6 contain the TET-labeled oligonucleotides 79. Lanes 1 and 4 contain products of reactions which were not heat treated. Lanes 2 and 5 contain products from reactions which were heat treated and lanes 3 and 6 contain products from reactions which were heat treated and subjected to phosphatase treatment.

As shown in Fig. 55, heat treatment causes significant breakdown of the labeled DNA, generating a ladder of degradation products (Fig. 55, lanes 2, 3, 5 and 6).

Band intensities correlate with purine and pyrimidine base positioning in the olig6nucleotide sequences, indicating that backbone hydrolysis may occur through formation of abasic intermediate products that have faster rates for purines then for pyrimidines (Lindahl and Karlstr6m, Biochem., 12:5151 [1973]).

Dephosphorylation decreases the mobility of all products generated by the thermal degradation process, with the most pronounced effect observed for the shorter products (Fig. 55, lanes 3 and This demonstrates that thermally degraded products possess a 3' end terminal phosphoryl group which can be removed by dephosphorylation with CIAP. Removal of the phosphoryl group decreases the overall negative charge by 2. Therefore, shorter products which have a small number of negative charges are influenced to a greater degree upon the removal of two charges. This leads to a larger mobility shift in the shorter products than that observed for the larger species.

The fact that the majority of thermally degraded DNA products contain 3' end phosphate groups and Cleavase® enzyme-generated products do not allowed the development of simple isolation methods for products generated in the invader-directed cleavage assay.

The extra two charges found in thermal breakdown products do not exist in the specific cleavage products. Therefore, if one designs assays that produce specific products which contain a net positive charge of one or two, then similar thermal breakdown products will either be negative or neutral. The difference can be used to isolate specific products by reverse charge methods as shown below.

b) Dephosphorylation Of Short Amino-Modified Oligonucleotides Can Reverse The Net Charge Of The Labeled Product To demonstrate how oligonucleotides can be transformed from net negative to net positively charged compounds, the four short amino-modified oligonucleotides labeled 70, 74, 75 and 76 and shown in Figs. 56-58 were synthesized (Fig. 56 shows both oligonucleotides and 74). All four modified oligonucleotides possess Cy-3 dyes positioned at the which individually are positively charged under reaction and isolation conditions described in 163 this example. Compounds 70 and 74 contain two amino modified thymidines that, under reaction conditions, display positively charged R-NH3' groups attached at the C5 position through a or C 6 linker, respectively. Because compounds 70 and 74 are 3'-end phosphorylated, they consist of four negative charges and three positive charges. Compound 75 differs from 74 in that the internal C 6 amino modified thymidine phosphate in 74 is replaced by a thymidine methyl phosphonate. The phosphonate backbone is uncharged and so there are a total of three negative charges on compound 75. This gives compound 75 a net negative one charge. Compound 76 differs from 70 in that the internal amino modified thymidine is replaced by an internal cytosine phosphonate. The pK, of the N3 nitrogen of cytosine can be from 4 to 7. Thus, the net charges of this compound, can be from -1 to 0 depending on the pH of the solution. For the simplicity of analysis, each group is assigned a whole number of charges, although it is realized that, depending on the pK, of each chemical group and ambient pH, a real charge may differ from the whole number assigned. It is assumed that this difference is not significant over the range of pHs used in the enzymatic reactions studied here.

Dephosphorylation of these compounds, or the removal of the 3' end terminal phosphoryl group, results in elimination of two negative charges and generates products that have a net positive charge of one. In this experiment, the method of isoelectric focusing (IEF) was used to demonstrate a change from one negative to one positive net charge for the described substrates during dephosphorylation.

Substrates 70, 74, 75 and 76 were synthesized by standard phosphoramidite chemistries and deprotected for 24 hours at 22 0 C in 14 M aqueous ammonium hydroxide solution, after which the solvent was removed in vacuo. The dried powders were resuspended in 200 pl of

SH

2 0 and filtered through 0.2 pnm filters. The concentration of the stock solutions was S 25 estimated by UV-absorbance at 261 nm of samples diluted 200-fold in H 2 0 using a spectrophotometer (Spectronic Genesys 2, Milton Roy).

Dephosphorylation of compounds 70 and 74, 75 and 76 was accomplished by treating 10 pi of the crude stock solutions (ranging in concentration from approximately 0.5 to 2 mM) with 2 units of CIAP in 100 pl of CIAP buffer (Promega) at 37 0 C for 1 hour. The reactions 30 were then heated to 75°C for 15 min. in order to inactivate the CIAP. For clarity, dephosphorylated compounds are designated For example, after dephosphorylation, substrate 70 becomes To prepare samples for IEF experiments, the concentration of the stock solutions of 164substrate and dephosphorylated product were adjusted to a uniform absorbance of 8.5 x 10' 3 at 532 nm by dilution with water. Two microliters of each sample were analyzed by IEF using a PhastSystem electrophoresis unit (Pharmacia) and PhastGel IEF 3-9 media (Pharmacia) according to the manufacturer's protocol. Separation was performed at 15°C with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75 Vh; load; 200 V, 2.5 mA. 3.5 W, Vh; run; 2,000 V; 2.5 mA; 3.5 W, 130 Vh. After separation, samples were visualized by using the FMBIO Image Analyzer (Hitachi) fitted with a 585 nm filter. The resulting imager scan is shown in Fig. 59.

Fig. 59 shows results of IEF separation of substrates 70, 74, 75 and 76 and their dephosphorylated products. The arrow labeled "Sample Loading Position" indicates a loading line, the sign shows the position of the positive electrode and the sign indicates the position of the negative electrode.

The results shown in Fig. 59 demonstrate that substrates 70, 74, 75 and 76 migrated toward the positive electrode, while the dephosphorylated products 70dp, 74dp, 75dp and 76dp migrated toward negative electrode. The observed differences in mobility direction was in accord with predicted net charge of the substrates (minus one) and the products (plus one).

Small perturbations in the mobilities of the phosphorylated compounds indicate that the overall pis vary. This was also true for the dephosphorylated compounds. The presence of the cytosine in 76dp, for instance, moved this compound further toward the negative electrode which was indicative of a higher overall pi relative to the other dephosphorylated compounds.

It is important to note that additional positive charges can be obtained by using a combination of natural amino modified bases (70dp and 74dp) along with uncharged methylphosphonate bridges (products 75dp and 76dp).

The results shown above demonstrate that the removal of a single phosphate group can 25 flip the net charge of an oligonucleotide to cause reversal in an electric field, allowing easy separation of products, and that the precise base composition of the oligonucleotides affect absolute mobility but not the charge-flipping effect.

*00**0 165 EXAMPLE 24 Detection Of Specific Cleavage Products In The Invader-Directed Cleavage Reaction By Charge Reversal In this examnle the ahilitvy isolat nArodct genPerate in the inver-irected cleavage assay from all other nucleic acids present in the reaction cocktail was demonstrated using charge reversal. This experiment utilized the following Cy3-labeled oligonucleotide: 5'-Cy3-AminoT-AminoT-CTTTTCACCAGCGAGACGGG-3' (SEQ ID NO:61; termed "oligonucleotide Oligonucleotide 61 was designed to release upon cleavage a net positively charged labeled product. To test whether or not a net positively charged labeled product would be recognized by the Cleavase® enzymes in the invader-directed cleavage assay format, probe oligonucleotide 61 (SEQ ID NO:61) and invading oligonucleotide 67 (SEQ ID NO:62) were chemically synthesized on a DNA synthesizer (ABI 391) using standard phosphoramidite chemistries and reagents obtained from Glen Research (Sterling, VA).

Each assay reaction comprised 100 fmoles of M13mpl8 single stranded DNA, pmoles each of the probe (SEQ ID NO:61) and invader (SEQ ID NO:62) oligonucleotides, and 20 units of Cleavase® A/G in a 10 pl solution of 10 mM MOPS, pH 7.4 with 100 mM KCI. Samples were overlaid with mineral oil to prevent evaporation. The samples were brought to either 50 0 C, 55 0 C, 60 0 C, or 65 0 C and cleavage was initiated by the addition of 1 e C pl of 40 mM MnCI 2 Reactions were allowed to proceed for 25 minutes and then were terminated by the addition of 10 pl of 95% formamide containing 20 mM EDTA and 0.02% methyl violet. The negative control experiment lacked the target M13mpl8 and was run at 0 C. Five microliters of each reaction were loaded into separate wells of a 20% denaturing polyacrylamide gel (cross-linked 29:1) with 8 M urea in a buffer containing 45 mM Tris- Borate (pH 8.3) and 1.4 mM EDTA. An electric field of 20 watts was applied for minutes, with the electrodes oriented as indicated in Fig. 60B in reverse orientation).

The products of these reactions were visualized using the FMBIO fluorescence imager and the resulting imager scan is shown in Fig. Fig. 60A provides a schematic illustration showing an alignment of the invader (SEQ ID NO:61) and probe (SEQ ID NO:62) along the target M13mpl8 DNA; only 53 bases of the M13mpl8 sequence is shown (SEQ ID NO:63). The sequence of the invader oligonucleotide is displayed under the M13mpl8 target and an arrow is used above the Ml3mpl8 sequence to indicate the position of the invader relative to the probe and target. As shown in Fig. 166the invader and probe oligonucleotides share a 2 base region of overlap.

In Fig. 60B, lanes 1-6 contain reactions performed at 50 0 C, 55 0 C, 60 0 C, and 65 0

C,

respectively; lane 5 contained the control reaction (lacking target). In Fig. 60B, the products of cleavage are seen as dark bands in the upper half of the panel; the faint lower band seen appears in proportion to the amount of primary product produced and, while not limiting the invention to a particular mechanism, may represent cleavage one nucleotide into the duplex.

The uncleaved probe does not enter the gel and is thus not visible. The control lane showed no detectable signal over background (lane As expected in an invasive cleavage reaction, the rate of accumulation of specific cleavage product was temperature-dependent. Using these particular oligonucleotides and target, the fastest rate of accumulation of product was observed at 55 0 C (lane 2) and very little product observed at 65 0 C (lane 4).

When incubated for extended periods at high temperature, DNA probes can break nonspecifically suffer thermal degradation) and the resulting fragments contribute an interfering background to the analysis. The products of such thermal breakdown are distributed from single-nucleotides up to the full length probe. In this experiment, the ability of charge based separation of cleavage products charge reversal) would allow the sensitive separation of the specific products of target-dependent cleavage from probe fragments generated by thermal degradation was examined.

To test the sensitivity limit of this detection method, the target M13mpl8 DNA was 20 serially diluted ten fold over than range of 1 fmole to 1 amole. The invader and probe oligonucleotides were those described above SEQ ID NOS:61 and 62). The invasive cleavage reactions were run as described above with the following modifications: the reactions were performed at 55 0 C, 250 mM or 100 mM KGlu was used in place of the 100 mM KCI and only 1 pmole of the invader oligonucleotide was added. The reactions were 25 initiated as described above and allowed to progress for 12.5 hours. A negative control reaction which lacked added M13m18 target DNA was also run. The reactions were terminated by the addition of 10 pl of 95% formamide containing 20 mM EDTA and 0.02% o •methyl violet, and 5 pl of these mixtures were electrophoresed and visualized as described above. The resulting imager scan is shown in Fig. 61.

In Fig. 61, lane 1 contains the negative control; lanes 2-5 contain reactions performed using 100 mM KGlu; lanes 6-9 contain reactions performed using 250 mM KGlu. The reactions resolved in lanes 2 and 6 contained 1 fmole of target DNA; those in lanes 3 and 7 contained 100 amole of target; those in lanes 4 and 8 contained 10 amole of target and those 167 in lanes 5 and 9 contained 1 amole of target. The results shown in Fig. 61 demonstrate that the detection limit using charge reversal to detect the production of specific cleavage products in an invasive cleavage reaction is at or below 1 attomole or approximately 6.02 x 10' target molecules. No detectable signal was observed in the control lane, which indicates that nonspecific hydrolysis or other breakdown products do not migrate in the same direction as enzyme-specific cleavage products. The excitation and emission maxima for Cy3 are 554 and 568, respectively, while the FMBIO Imager Analyzer excites at 532 and detects at 585.

Therefore, the limit of detection of specific cleavage products can be improved by the use of more closely matched excitation source and detection filters.

EXAMPLE Devices And Methods For The Separation And Detection Of Charged Reaction Products This example is directed at methods and devices for isolating and concentrating specific reaction products produced by enzymatic reactions conducted in solution whereby the reactions generate charged products from either a charge neutral substrate or a substrate eee e bearing the opposite charge borne by the specific reaction product. The methods and devices of this example allow isolation of, for example, the products generated by the invader-directed cleavage assay of the present invention.

.20 The methods and devices of this example are based on the principle that when an electric field is applied to a solution of charged molecules, the migration of the molecules toward the electrode of the opposite charge occurs very rapidly. If a matrix or other inhibitory material is introduced between the charged molecules and the electrode of opposite charge such that this rapid migration is dramatically slowed, the first molecules to reach the matrix will be nearly stopped, thus allowing the lagging molecules to catch up. In this way a eeee dispersed population of charged molecules in solution can be effectively concentrated into a smaller volume. By tagging the molecules with a detectable moiety a fluorescent dye), °detection is facilitated by both the concentration and the localization of the analytes. This example illustrates two embodiments of devices contemplated by the present invention; of course, variations of these devices will be apparent to those skilled in the art and are within the spirit and scope of the present invention.

168 Fig. 62 depicts one embodiment of a devic lfor concentrating the positively-charged products generated using the methods of the present invention. As shown in Fig. 62, the device comprises a reaction tube (10) which contains the reaction solution One end of pach nf thun thin rnnillqriPe? n r olher tnhes with a hollow core) (13A and 1l3R) are suhmpered in the reaction solution The capillaries (13A and 13B) may be suspended in the reaction solution (11) such that they are not in contact with the reaction tube itself; one appropriate method of suspending the capillaries is to hold them in place with clamps (not shown).

Alternatively, the capillaries may be suspended in the reaction solution (11) such that they are in contact with the reaction tube itself. Suitable capillaries include glass capillary tubes commonly available from scientific supply companies Fisher Scientific or VWR Scientific) or from medical supply houses that carry materials for blood drawing and analysis.

Though the present invention is not limited to capillaries of any particular inner diameter, tubes with inner diameters of up to about 1/8 inch (approximately 3 mm) are particularly preferred for use with the present invention; for example Kimble No. 73811-99 tubes (VWR Scientific) have an inner diameter of 1.1 mm and are a suitable type of capillary tube.

Although the capillaries of the device are commonly composed of glass, any nonconductive tubular material, either rigid or flexible, that can contain either a conductive material or a trapping material is suitable for use in the present invention. One example of a suitable flexible tube is Tygon® clear plastic tubing (Part No. R3603; inner diameter 1/16 inch; 20 outer diameter 1/8 inch).

As illustrated in Fig. 62, capillary 13A is connected to the positive electrode of a power supply (20) a controllable power supply available through the laboratory suppliers listed above or through electronics supply houses like Radio Shack) and capillary 13B is connected to the negative electrode of the power supply Capillary 13B is filled with a trapping material (14) capable of trapping the positively-charged reaction products by e allowing minimal migration of products that have entered the trapping material Suitable trapping materials include, but are not limited to, high percentage about acrylamide polymerized in a high salt buffer (0.5 M or higher sodium acetate or similar salt); such a high percentage polyacrylamide matrix dramatically slows the migration of the positively-charged reaction products. Alternatively, the trapping material may comprise a solid, negatively-charged matrix, such as negatively-charged latex beads, that can bind the incoming positively-charged products. It should be noted that any amount of trapping material (14) capable of inhibiting any concentrating the positively-charged reaction products 169 may be used. Thus, while the capillary 13B in Fig. 62 only contains trapping material in the lower, submerged portion of the tube, the trapping material (14) can be present in the entire capillary (13B); similarly, less trapping material (14) could be present than that shown in Fig. 62 because the positively-charged reaction products generally accumulate within a very small portion ol the bottom of the capillary (13B). The amount of trapping material need only be sufficient to make contact with the reaction solution (11) and have the capacity to collect the reaction products. When capillary 13B is not completely filled with the trapping material, the remaining space is filled with any conductive material suitable conductive materials are discussed below.

By comparison, the capillary (13A) connected to the positive electrode of the power supply 20 may be filled with any conductive material (15; indicated by the hatched lines in Fig. 62). This may be the sample reaction buffer 10 mM MOPS, pH 7.5 with 150 mM LiCI, 4 mM MnCI,), a standard electrophoresis buffer 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA), or the reaction solution (11) itself. The conductive material (15) is frequently a liquid, but a semi-solid material a gel) or other suitable material might be easier to use and is within the scope of the present invention. Moreover, that trapping material used in the other capillary capillary 13B) may also be used as the conductive material. Conversely, it should be noted that the same conductive material used in the capillary (13A) attached to the positive electrode may also be used in capillary 13B to fill the space above the region 20 containing the trapping material (14) (see Fig. 62).

The top end of each of the capillaries (13A and 13B) is connected to the appropriate electrode of the power supply (20) by electrode wire (18) or other suitable material. Fine platinum wire 0.1 to 0.4 mm, Aesar Johnson Matthey, Ward Hill, MA) is commonly used as conductive wire because it does not corrode under electrophoresis conditions. The electrode wire (18) can be attached to the capillaries (13A and 13B) by a nonconductive adhesive (not shown), such as the silicone adhesives that are commonly sold in hardware stores for sealing plumbing fixtures. If the capillaries are constructed of a flexible material, the electrode wire (18) can be secured with a small hose clamp or constricting wire (not shown) to compress the opening of the capillaries around the electrode wire. If the conducting material (15) is a gel, an electrode wire (18) can be embedded directly in the gel within the capillary.

170- The cleavage reaction is assembled in the reaction tube (10) and allowed to proceed therein as described in proceeding examples Examples 22-23). Though not limited to any particular volume of reaction solution a preferred volume is less than 10 ml and more preferably less than 0.1 ml. The volume need only be sufficient to permit contact with both capillaries. After the cleavage reaction is completed, an electric field is applied to the capillaries by turning on the power source As a result, the positively-charged products generated in the course of the invader-directed cleavage reaction which employs an oligonucleotide, which when cleaved, generates a positively charged fragment (described in Ex. 23) but when uncleaved bears a net negative charge, migrate to the negative capillary, where their migration is slowed or stopped by the trapping material and the negativelycharged uncut and thermally degraded probe molecules migrate toward the positive electrode.

Through the use of this or a similar device, the positively-charged products of the invasive cleavage reaction are separated from the other material uncut and thermally degraded probe) and concentrated from a large volume. Concentration of the product in a small amount of trapping material (14) allows for simplicity of detection, with a much higher signal-to-noise ratio than possible with detection in the original reaction volume. Because the concentrated product is labeled with a detectable moiety like a fluorescent dye, a commercially-available fluorescent plate reader (not shown) can be used to ascertain the amount of product. Suitable plate readers include both top and bottom laser readers.

Capillary 13B can be positioned with the reaction tube (10) at any desired position so as to accommodate use with either a top or a bottom plate reading device.

In the alternative embodiment of the present invention depicted in Fig. 63, the procedure described above is accomplished by utilizing only a single capillary (13B). The capillary (13B) contains the trapping material (14) described above and is connected to an 25 electrode wire which in turn is attached to the negative electrode of a power supply The reaction tube (10) has an electrode (25) embedded into its surface such that one surface of the electrode is exposed to the interior of the reaction tube (10) and another surface is i exposed to the exterior of the reaction tube. The surface of the electrode (25) on the exterior of the reaction tube is in contact with a conductive surface (26) connected to the positive electrode of the power supply (20) through an electrode wire Variations of the arrangement depicted in Fig. 63 are also contemplated by the present invention. For example, the electrode (25) may be in contact with the reaction solution (11) through the use of a small 171 hole in the reaction tube furthermore, the electrode wire (18) can be directly attached to the electrode wire thereby eliminating the conductive surface (26).

As indicated in Fig. 63, the electrode (25) is embedded in the bottom of a reaction tube (10) such that one or more reaction tubes may be set on the conductive surface (26).

This conductive surface could serve as a negative electrode for multiple reaction tubes; such a surface with appropriate contacts could be applied through the use of metal foils copper or platinum, Aesar Johnson Matthey, Ward Hill, MA) in much the same way contacts are applied to circuit boards. Because such a surface contact would not be exposed to the reaction sample directly, less expensive metals, such as the copper could be used to make the electrical connections.

The above devices and methods are not limited to separation and concentration of positively charged oligonucleotides. As will be apparent to those skilled in the art, negatively charged reaction products may be separated from neutral or positively charged reactants using the above device and methods with the exception that capillary 13B is attached to the positive electrode of the power supply (20) and capillary 13A or alternatively, electrode 25, is attached to the negative electrode of the power supply o.

"EXAMPLE 26 Primer-Directed And Primer Independent Cleavage 20 Occur At The Same Site When The Primer Extends To The 3' Side Of A Mismatched "Bubble" In The Downstream Duplex As discussed above in Ex. 1, the presence of a primer upstream of a bifurcated duplex .can influence the site of cleavage, and the existence of a gap between the 3' end of the primer and the base of the duplex can cause a shift of the cleavage site up the unpaired 5' arm of the 25 structure (see also Lyamichev et al., supra and U.S. Patent No. 5,422,253). The resulting non-invasive shift of the cleavage site in response to a primer is demonstrated in Figs. 9, and 1 1, in which the primer used left a 4-nucleotide gap (relative to the base of the duplex).

S: ~In Figs. 9-11, all of the "primer-directed" cleavage reactions yielded a 21 nucleotide product, while the primer-independent cleavage reactions yielded a 25 nucleotide product. The site of cleavage obtained when the primer was extended to the base of the duplex, leaving no gap was examined. The results are shown in Fig. 64 (Fig. 64 is a reproduction of Fig. 2C in Lyamichev et al. These data were derived from the cleavage of the structure shown in Fig. 6, as described in Ex. 1. Unless otherwise specified, the cleavage reactions comprised 0.01 -172 pmoles of heat-denatured, end-labeled hairpin DNA (with the unlabeled complementary strand also present), 1 pmole primer [complementary to the 3' arm shown in Fig. 6 and having the sequence: 5'-GAAT TCGATTTAGGTGACACTATAGAATACA (SEQ ID NO:64)] and units of DNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 pl of 10 mM Tris- Cl, pH 8.5, and 1.5 mM MgCl 2 and 50 mM KCI. The primer was omitted from the reaction shown in the first lane of Fig. 64 and included in lane 2. These reactions were incubated at for 10 minutes. Reactions were initiated at the final reaction temperature by the addition of either the MgCI 2 or enzyme. Reactions were stopped at their incubation temperatures by the addition of 8 .l of 95% formamide with 20 mM EDTA and 0.05% marker dyes.

Fig. 64 is an autoradiogram that indicates the effects on the site of cleavage of a bifurcated duplex structure in the presence of a primer that extends to the base of the hairpin duplex. The size of the released cleavage product is shown to the left 25 nucleotides).

A dideoxynucleotide sequencing ladder of the cleavage substrate is shown on the right as a marker (lanes 3-6).

These data show that the presence of a primer that is adjacent to a downstream duplex (lane 2) produces cleavage at the same site as seen in reactions performed in the absence of the primer (lane 1) (see Figs. 9A and B, 10B and 11A for additional comparisons). When the 3' terminal nucleotides of the upstream oligonucleotide can base pair to the template strand but are not homologous to the displaced strand in the region immediately upstream of the cleavage site when the upstream oligonucleotide is opening up a "bubble" in the duplex), the site to which cleavage is apparently shifted is not wholly dependent on the presence of an upstream oligonucleotide.

As discussed above in the Background section and in Table 1, the requirement that 25 two independent sequences be recognized in an assay provides a highly desirable level of specificity. In the invasive cleavage reactions of the present invention, the invader and probe oligonucleotides must hybridize to the target nucleic acid with the correct orientation and spacing to enable the production of the correct cleavage product. When the distinctive pattern of cleavage is not dependent on the successful alignment of both oligonucleotides in the detection system these advantages of independent recognition are lost.

173 EXAMPLE 27 Invasive Cleavage And Primer-Directed Cleavage When There Is Only Partial Homology In The Overlap Region While not limiting the present invention to any particular mechanism, invasive cleavage occurs when the site of cleavage is shifted to a site within the duplex formed between the probe and the target nucleic acid in a manner that is dependent on the presence of an upstream oligonucleotide which shares a region of overlap with the downstream probe oligonucleotide. In some instances, the 5' region of the downstream oligonucleotide may not be completely complementary to the target nucleic acid. In these instances, cleavage of the probe may occur at an internal site within the probe even in the absence of an upstream oligonucleotide (in contrast to the base-by-base nibbling seen when a fully paired probe is used without an invader). Invasive cleavage is characterized by an apparent shifting of cleavage to a site within a downstream duplex that is dependent on the presence of the invader oligonucleotide.

A comparison between invasive cleavage and primer-directed cleavage may be illustrated by comparing the expected cleavage sites of a set of probe oligonucleotides having decreasing degrees of complementarity to the target strand in the 5' region of the probe the region that overlaps with the invader). A simple test, similar to that performed on the hairpin substrate above (Ex. 25), can be performed to compare invasive cleavage with the S 20 non- invasive primer-directed cleavage described above. Such a set of test oligonucleotides is diagrammed in Fig. 65. The structures shown in Fig. 65 are grouped in pairs, labeled and Each pair has the same probe sequence annealed to the target strand (SEQ ID NO:65), but the top structure of each pair is drawn without an upstream oligonucleotide, while the bottom structure includes this oligonucleotide (SEQ ID NO:66). The sequences of 25 the probes shown in Figs. 64a-64d are listed in SEQ ID NOS:43, 67, 68 and 69, respectively.

Probable sites of cleavage are indicated by the black arrowheads. (It is noted that the precise site of cleavage on each of these structures may vary depending on the choice of cleavage agent and other experimental variables. These particular sites are provided for illustrative purposes only.) To conduct this test, the site of cleavage of each probe is determined both in the presence and the absence of the upstream oligonucleotide, in reaction conditions such as those described in Ex. 19. The products of each pair of reactions are then be compared to 174determine whether the fragment released from the 5' end of the probe increases in size when the upstream oligonucleotide is included in the reaction.

The arrangement shown in Fig. 65a, in which the probe molecule is completely cmpll -met*" the th target strandr is similar to that shown in Fig. 32. Treatment of the top structure with the 5' nuclease of a DNA polymerase would cause exonucleolytic nibbling of the probe in the absence of the upstream oligonucleotide). In contrast, inclusion of an invader oligonucleotide would cause a distinctive cleavage shift similar, to those observed in Fig. 33.

The arrangements shown in Figs. 65b and 65c have some amount of unpaired sequence at the 5' terminus of the probe 3 and 5 bases, respectively). These small 5' arms are suitable cleavage substrate for the 5' nucleases and would be cleaved within 2 nucleotide's of the junction between the single stranded region and the duplex. In these arrangements, the 3' end of the upstream oligonucleotide shares identity with a portion of the 5' region of the probe which is complementary to the target sequence (that is the 3' end of the invader has to compete for binding to the target with a portion of the 5' end of the probe). Therefore, when the upstream oligonucleotide is included it is thought to mediate a shift in the site of cleavage into the downstream duplex (although the present invention is not limited to any particular o mechanism of action), and this would, therefore, constitute invasive cleavage. If the extreme 5' nucleotides of the unpaired region of the probe were able to hybridize to the target strand, 20 the cleavage site in the absence of the invader might change but the addition of the invader oligonucleotide would still shift the cleavage site to the proper position.

Finally, in the arrangement shown in Fig. 65d, the probe and upstream oligonucleotides share no significant regions of homology, and the presence of the upstream oligonucleotide would not compete for binding to the target with the probe. Cleavage of the 25 structures shown in Fig. 64d would occur at the same site with or without the upstream oligonucleotide, and is thus would not constitute invasive cleavage.

By examining any upstream oligonucleotide/probe pair in this way, it can easily be determined whether the resulting cleavage is invasive or merely primer-directed. Such analysis is particularly useful when the probe is not fully complementary to the target nucleic acid, so that the expected result may not be obvious by simple inspection of the sequences.

175 EXAMPLE 28 Modified Cleavase® Enzymes In order to develop nucleases having useful activities for the cleavage of nucleic acids the following modified nucleases were produced.

a) Cleavase® BIN/ihruimbin Nuciease i) Cloning and Expression of Cleavase® BN/thrombin Nuclease Site-directed mutagenesis was used to introduce a protein sequence recognized by the protease thrombin into the region of the Cleavase® BN nuclease which is thought to form the helical arch of the protein through which the single-stranded DNA that is cleaved must presumably pass. Mutagenesis was carried out using the TransformerTM mutagenesis kit (Clonetech) according to manufacturer's protocol using the mutagenic oligonucleotide CGGGACGAGCGTGGGGGCCCG (SEQ ID NO:100).

After mutagenesis, the DNA was sequenced to verify the insertion of the thrombin cleavage site. The DNA sequence encoding the Cleavase® BN/thrombin nuclease is provided in SEQ ID NO:101; the amino acid sequence of Cleavase® BN/thrombin nuclease is provided in SEQ ID NO:102.

A large scale preparation of the thrombin mutant Cleavase® BN/thrombin) was done using E. coli cells overexpressing the Cleavase® BN/thrombin nuclease as described in Ex. 28.

20 ii) Thrombin Cleavage of Cleavase® BN/thrombin Six point four mg of the purified Cleavase® BN/thrombin nuclease was digested with 0.4 U of thrombin (Novagen, Madison, WI) for 4 hours at 23°C or 37 0 C. Complete ~digestion was verified by electrophoresis on a 15% SDS polyacrylamide gel followed by staining with Coomassie Brilliant Blue R. Wild-type Cleavase® BN nuclease was also digested with thrombin as a control. The resulting gel is shown in Fig. 72.

In Fig. 72, lane 1 contains molecular weight markers (Low-Range Protein Molecular Weight Markers; Promega), lane 2 contains undigested Cleavase® BN/thrombin nuclease, lanes 3 and 4 contain Cleavase® BN/thrombin nuclease digested with thrombin at 23 0 C for 2 and 4 hours, respectively, and lanes 5 and 6 contain Cleavase® BN/thrombin nuclease digested with thrombin at 37 0 C for 2 and 4 hours, respectively. These results show that the Cleavase® BN/thrombin nuclease has an apparent molecular weight of 36.5 kilodaltons and demonstrate that Cleavase® BN/thrombin nuclease is efficiently cleaved by thrombin. In addition, the thrombin cleavage products have approximate molecular weights of 27 176kilodaltons and 9 kilodaltons, the size expected based upon the position of the inserted thrombin site in the Cleavase® BN/thrombin nuclease.

To determine the level of hairpin cleavage activity in digested and undigested Cleavase® BN/thrombin nuclease, dilutions were made and used to cleave a test hairoin containing a 5' fluorescein label. Varying amounts of digested and undigested Cleavase® BN/thrombin nuclease were incubated with 5 pM oligonucleotide S-60 hairpin (SEQ ID see Fig. 30) in 10 mM MOPS (pH 0.05% Tween-20, 0.05% NP-40, and 1 mM MnCI1 for 5 minutes at 60 0 C. The digested mixture was electrophoresed on a acrylamide gel and visualized on a Hitachi FMBIO 100 fluorescence imager. The resulting image is shown in Fig. 73.

In Fig. 73, lane 1 contains the no enzyme control, lane 2 contains reaction products produced using 0.01 ng of Cleavase® BN nuclease, lanes 3, 4, and 5 contain reaction products produced using 0.01 ng, 0.04 ng, and 4 ng of undigested Cleavase® BN/thrombin nuclease, respectively, and lanes 6, 7, and 8 contain reaction products produced using 0.01 ng, 0.04 ng, and 4 ng of thrombin-digested Cleavase® BN/thrombin nuclease, respectively. The results shown in Fig. 73 demonstrated that the insertion of the thrombin cleavage site reduced cleavage activity about 200-fold (relative to the activity of Cleavase® BN nuclease), but that digestion with thrombin did not reduce the activity significantly.

MI3 single-stranded DNA was used as a substrate for cleavage by Cleavase® BN nuclease and digested and undigested Cleavase® BN/thrombin nuclease. Seventy nanograms of single-stranded M13 DNA (New England Biolabs, Beverly, MA) was incubated in 10 mM MOPS, pH 7.5, 0.05% Tween-20, 0.05% NP-40, 1 mM MgCI 2 or 1 mM MnCl,, with 8 ng of **Cleavase® BN nuclease, undigested Cleavase® BN/thrombin nuclease, or digested Cleavase® BN/thrombin nuclease for 10 minutes at 50 0 C. Reaction mixtures were electrophoresed on a 25 0.8% agarose gel and then stained with a solution containing 0.5 Pg/ml ethidium bromide (EtBr) to visualize DNA bands. A negative image of the EtBr-stained gel is shown in Fig.

74.

In Fig. 74, lane 1 contains the no enzyme control, lane 2 contains reaction products produced using Cleavase® BN nuclease and 1 mM MgC 2 lane 3 contains reaction products produced using Cleavase® BN nuclease and 1 mM MnC1 2 lane 4 contains reaction products produced using undigested Cleavase® BN/thrombin nuclease and 1 mM MgCl,, lane contains reaction products produced using undigested Cleavase® BN/thrombin nuclease and 1 mM MnCl 2 lane 6 contains reaction products produced using thrombin-digested Cleavase® 177 BN/thrombin nuclease and 1 mM MgCI 2 and lane 7 contains reaction products produced using thrombin-digested Cleavase® BN/thrombin nuclease and 1 mM MnCI 2 The results shown in Fig. 74 demonstrated that the Cleavase® BN/thrombin nuclease had an enhanced ability t cleave circular DNA (and thus a reduced requirement for the presence of a free end) as compared to the Cleavase® BN nuclease.

It can be seen from these data that the helical arch of these proteins can be opened without destroying the enzyme or its ability to specifically recognize cleavage structures. The Cleavase® BN/thrombin mutant has an increased ability to cleave without reference to a end, as discussed above. The ability to cleave such structures will allow the cleavage of long molecules, such as genomic DNA that, while often not circular, may present many desirable cleavage sites that are at a far removed from any available 5' end. Cleavage structures may be made at such sites either by folding of the strands CFLP® cleavage) or by the introduction of structure-forming oligonucleotides Patent No. 5,422,253). 5' ends of nucleic acids can also be made unavailable because of binding of a substance too large to thread through the helical arch. Such binding moieties may include proteins such as streptavidin or antibodies, or solid supports such as beads or the walls of a reaction vessel. A cleavage enzyme with an opening in the loop of the helical arch will be able to cleave DNAs that are configured in this way, extending the number of ways in which reactions using such enzymes can be formatted.

20 b) Cleavase® DN Nuclease i) Construction and Expression of Cleavase® DN Nuclease A polymerization deficient mutant of Taq DNA polymerase, termed Cleavase® DN nuclease. was constructed. Cleavase® DN nuclease contains an asparagine residue in place of the wild-type aspartic acid residue at position 785 (D785N).

25 DNA encoding the Cleavase® DN nuclease was constructed from the gene encoding for Cleavase® A/G (mutTaq, Ex. 2) in two rounds of site-directed mutagenesis. First, the G at position 1397 and the G at position 2264 of the Cleavase® A/G gene (SEQ ID NO:21) were changed to A at each position to recreate a wild-type DNAPTaq gene. As a second round of mutagenesis, the wild type DNAPTaq gene was converted to the Cleavase® DN gene by changing the G at position 2356 to A. These manipulations were performed as follows.

178 DNA encoding the Cleavase® A/G nuclease was recloned from pTTQ18 plasmid (Ex.

2) into the pTrc99A plasmid (Pharmacia) in a two step procedure. First, the pTrc99A vector was modified by removing the G at position 270 of the pTrc99A map, creating the pTrc99G cloning vecLui. To t:his e, pTrc99A p.asmd DNA was cut with NcoI and the recessive 3' ends were filled-in using the Klenow fragment of E.coli polymerase I in the presence of all four dNTPs at 37°C for 15 min. After inactivation of the Klenow fragment by incubation at 0 C for 10 min, the plasmid DNA was cut with EcoRI, the ends were again filled-in using the Klenow fragment in the presence of all four dNTPs at 37 0 C for 15 min. The Klenow fragment was then inactivated by incubation at 65 0 C for 10 min. The plasmid DNA was ethanol precipitated, recircularized by ligation, and used to transform E.coli JM109 cells (Promega). Plasmid DNA was isolated from single colonies and deletion of the G at position 270 of the pTrc99A map was confirmed by DNA sequencing.

As a second step, DNA encoding the Cleavase® A/G nuclease was removed from the pTTQ18 plasmid using EcoRI and Sail and the DNA fragment carrying the Cleavase® A/G nuclease gene was separated on a 1% agarose gel and isolated with Geneclean II Kit (Bio 101, Vista. CA). The purified fragment was ligated into the pTrc99G vector which had been cut with EcoRI and Sail. The ligation mixture was used to transform competent E.coli JM109 cells (Promega). Plasmid DNA was isolated from single colonies and insertion of the o Cleavase® A/G nuclease gene was confirmed by restriction analysis using EcoRI and Sail.

20 Plasmid DNA pTrcAG carrying the Cleavase® A/G nuclease gene cloned into the pTrc99A vector was purified from 200 ml of JM109 overnight culture using QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, CA) according to manufacturer's protocol. pTrcAG plasmid DNA was mutagenized using two mutagenic primers, E465 (SEQ ID NO:103) (Integrated DNA Technologies, Iowa) and R754Q (SEQ ID NO:104) (Integrated DNA 25 Technologies), and the selection primer Trans Oligonucleotide AlwNI/Spel (Clontech, Palo Alto, CA, catalog #6488-1) according to Transformer M Site-Directed Mutagenesis Kit protocol (Clontech, Palo Alto, CA) to produce a restored wild-type DNAPTaq gene (pTrcWT).

S* pTrcWT plasmid DNA carrying the wild-type DNAPTaq gene cloned into the pTrc99A vector was purified from 200 ml of JM109 overnight culture using QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, CA) according to manufacturer's protocol. pTrcWT was then mutagenized using the mutagenic primer D785N (SEQ ID NO:105) (Integrated DNA Technologies) and the selection primer Switch Oligonucleotide SpeI/AlwNI (Clontech, Palo 179- Alto, CA. catalog #6373-1) according to TransformerTM Site-Directed Mutagenesis Kit protocol (Clontech, Palo Alto, CA) to create a plasmid containing DNA encoding the Cleavase® DN nuclease. The DNA sequence encoding the Cleavase® DN nuclease is proided in SEQ ID NO:106; the amino acid sequence of Cleavase® DN nuclease is provided in SEQ ID NO:107.

A large scale preparation of the Cleavase® DN nuclease was done using E. coli cells overexpressing the Cleavase® DN nuclease as described in Ex. 29.

c) Cleavase® DA Nuclease and Cleavase® DV Nuclease Two polymerization deficient mutants of Taq DNA polymerase, termed Cleavase® DA nuclease and Cleavase® DV nuclease, were constructed. The Cleavase® DA nuclease contains a alanine residue in place of the wild-type aspartic acid residue at position 610 (D785A). The Cleavase® DV nuclease contains a valine residue in place of the wild-type aspartic acid residue at position 610 (D610V).

i) Construction and Expression of the Cleavase® DA and Cleavase® DV Nucleases To construct vectors encoding the Cleavase® DA and DV nucleases, the Cleavase® A/G nuclease gene contained within pTrcAG was mutagenized with two mutagenic primers, R754Q (SEQ ID NO:104) and D610AV (SEQ ID NO:128) and the selection primer Trans Oligonucleotide AlwNI/Spel (Clontech, catalog #6488-1) according to the TransformerTM Site-Directed Mutagenesis Kit protocol (Clontech,) to create a plasmid containing DNA encoding the Cleavase® DA nuclease or Cleavase® DV nuclease. The D610AV oligonucleotide was synthesized to have a purine, A or G, at position 10 from the 5' end of the oligonucleotide. Following mutagenesis, plasmid DNA was isolated from single colonies and the type of mutation present, DA or DV, was determined by DNA sequencing. The DNA 25 sequence encoding the Cleavase® DA nuclease is provided in SEQ ID NO:129; the amino acid sequence of Cleavase® DA nuclease is provided in SEQ ID NO:130. The DNA sequence encoding the Cleavase® DV nuclease is provided in SEQ ID NO:131; the amino acid sequence of Cleavase® DV nuclease is provided in SEQ ID NO:132.

Large scale preparations of the Cleavase® DA and Cleavase® DV nucleases was done using E. coli cells overexpressing the Cleavase® DA nuclease or the Cleavase® DV nuclease as described in Ex. 29.

180d) Cleavase® Tth DN Nuclease i) Construction and Expression of Cleavase® TthDN Nuclease The DNA polymerase enzyme from the bacterial species Thermus thermophilus (Tth) was produced by cloning the gene for this protein into an expression vector and overproducing it in E. coli cells. Genomic DNA was prepared from 1 vial of dried Thermus thermophilus strain HB-8 from ATCC (ATCC #27634) as described in Ex. 29a. The DNA polymerase gene was amplified by PCR as described in Ex. 29b using the following primers: 5'-CACGAATTCCGAGGCGATGCTTCCGCTC-3' (SEQ ID NO:166) and 5'-TCGACGTCGACTAACCCTTGGCGGAAAGCC-3' (SEQ ID NO:167),as described in Ex.

29a.

The resulting PCR product was digested with EcoR I and Sall restriction endonucleases and inserted into EcoRIlSal I digested plasmid vector pTrc99g (described in Example 27b) by ligation, as described in Example 27b, to create the plasmid pTrcTth-1.

This Tth polymerase construct is missing a single nucleotide which was inadvertently omitted from the 5' oligonucleotide, resulting in the polymerase gene being out of frame. This mistake was corrected by mutagenesis of pTrcTth-1 as described in Example 27b using the following oligonucleotide: 5'-GCATCGCCTCGGAATTCATGGTC-3' (SEQ ID NO:168), to o° create the plasmid pTrcTth-2. The Tth DN construct was created by mutating the sequence encoding an aspartic acid at position 787 to a sequence encoding asparagine. Mutagenesis of 20 pTrcTth-2 with the following oligonucleotide: 3' (SEQ ID NO:169) as described in Example 27b, to create the plasmid pTrcTth-DN. The resulting polymerase-deficient nuclease, Cleavase® TthDN was expressed and purified as described in Ex. 29.

25 EXAMPLE 29 -Cloning And Expression of Thermostable FEN-1 Endonucleases Sequences encoding thermostable FEN-1 proteins derived from three Archaebacterial species were cloned and overexpressed in E. coli. This Example involved: a) Cloning and Expression of a FEN-1 Endonuclease from Methanococcus jannaschii; b) Cloning and Expression of a FEN-1 Endonuclease from Pyrococcusfuriosus; c) Cloning and Expression of a FEN-1 Endonuclease from Pyrococcus woesei; d) Cloning and Expression of a FEN-1 Endonuclease from Archaeoglobus fulgidus; e) Cloning and Expression of a FEN-1 Endonuclease from Methanobacterium thermoautotrophicum: f) Large Scale Preparation of 181 Recombinant Thermostable FEN-1 Proteins; and e) Activity Assays using FEN-I endonucleases.

a) Cloning and Expression Of A FEN-1 Endonuclease From Methanococcus jannaschii In this Example, DNA encoding the FEN- endonuclease from Methanococcus jannaschii jannaschii) was isolated from M. jannaschii cells and inserted into a plasmid under the transcriptional control of an inducible promoter as follows. Genomic DNA was prepared from 1 vial of live M. jannaschii bacteria (DSMZ 2661) with the DNA XTRAX kit (Gull), according to the manufacturer's protocol. The final DNA pellet was resuspended in 100 pl of TE (10 mM Tris HCI, pH 8.0, 1 mM EDTA). One microliter of the DNA solution was employed in a PCR using the AdvantageTM cDNA PCR kit (Clonetech); the PCR was conducted according to manufacturer's recommendations. The 5'-end primer (SEQ ID NO:108) is complementary to the 5' end of the Mja FEN-1 open reading frame with a one base substitution to create an Ncol restriction site (a fragment of the M. jannaschii genome which contains the gene encoding M. jannaschii (Mja) FEN-1 is available from GenBank as accession U67585). The 3'-end primer (SEQ ID NO:109) is complementary to a sequence about 15 base pairs downstream from the 3' end of the Mja FEN-1 open reading frame with 2 i base substitutions to create a Sall restriction enzyme site. The sequences of the 5'-end and 3'-end primers are: 5'-GGGATACCATGGGAGTGCAGTTTGG-3' (SEQ ID NO:108) and 5'-GGTAAATTTTTCTCGTCGACATC CCAC-3' (SEQ ID NO:109), respectively. The PCR reaction resulted in the amplification production) of a single major band about 1 kilobase in length. The open reading frame (ORF) encoding the Mja FEN-1 endonuclease is provided in SEQ ID NO:110; the amino acid sequence encoded by this ORF is provided in SEQ ID NO:111.

25 Following the PCR amplification, the entire reaction was electrophoresed on a agarose gel and the major band was excised from the gel and purified using the Geneclean II kit (BiolOl, Vista, CA) according to manufacturer's instructions. Approximately 1 jpg of the gel-purified Mja FEN-1 PCR product was digested with Ncol and Sail. After digestion, the DNA was purified using the Geneclean II kit according to manufacturer's instructions. One microgram of the pTrc99a vector (Pharmacia) was digested with Ncol and Sail in preparation for ligation with the digested PCR product. One hundred nanograms of digested pTrc99a vector and 250 ng of digested Mja FEN-1 PCR product were combined and ligated to create 182 pTrc99-MJFEN1. pTrc99-MJFEN was used to transform competent E. coli JM109 cells (Promega) using standard techniques.

b) Cloning and Expression Of A FEN-1 Endonuclease From Pvrococcus furiosus DNA encoding the Pyrococcus furiosus furiosus) FEN-1 endonuclease was obtained by PCR amplification using a plasmid containing DNA encoding the P. furiosus (Pfu) FEN-1 endonuclease (obtained from Dr. Frank Robb, Center of Marine Biotechnology, Baltimore, MD). DNA sequences encoding a portion of the Pfu FEN-1 endonuclease can be obtained from GenBank (Accession Nos. AA113505 and W36094). The amplified Pfu FEN-1 gene was inserted into the pTrc99a expression vector (Pharmacia) to place the Pfu FEN-I gene under the transcriptional control of the inducible trc promoter. The PCR amplification was conducted as follows. One hundred microliter reactions contained 50 mM Tris HC1, pH 20 mM (NH 4 2

SO

4 2 mM MgCl 2 50 gM dNTPs, 50 pmole each primer, 1 U Tfl polymerase (Epicentre) and 1 ng of FEN-1 gene-containing plasmid DNA. The 5'-end primer (SEQ ID NO:112) is complementary to the 5' end of the Pfu FEN-1 open reading frame but with two substitutions to create an NcoI site and the 3'-end primer (SEQ ID NO:113) is complementary to a region located about 30 base pairs downstream of the FEN-1 open reading frame with two substitutions to create a PstI site. The sequences of the 5'-end and 3'-end primers are: 5'-GAGGTGATACCATGGGTGTCC-3' (SEQ ID NO:112) and 20 5'-GAAACTCTGCAGCGCGTCAG-3' (SEQ ID NO:113), respectively. The PCR reaction resulted in the amplification of a single major band about 1 kilobase in length. The open reading frame (ORF) encoding the Pfu FEN-1 endonuclease is provided in SEQ ID NO:114; the amino acid sequence encoded by this ORF is provided in SEQ ID NO:115.

Following the PCR amplification, the entire reaction was electrophoresed on a 25 agarose gel and the major band was excised from the gel and purified using the Geneclean II kit (BiolOl, Vista, CA) according to manufacturer's instructions. Approximately 1 jig of gel purified Pfu FEN-1 PCR product was digested with Ncol and PstI. After digestion, the DNA S* was purified using the Geneclean II kit according to manufacturer's instructions. One microgram of the pTrc99a vector was digested with Ncol and Pstl prior to ligation with the digested PCR product. One hundred nanograms of digested pTrc99a and 250 ng of digested Pfu FEN-1 PCR product were combined and ligated to create pTrc99-PFFEN1.

pTrc99-PFFEN1 was used to transform competent E. coli JM109 cells (Promega) using standard techniques.

183 c) Cloning and Expression Of A FEN-1 Endonuclcase From Pyrococcus woesei For the cloning of DNA encoding the Pyrococcus woesei (Pwo) FEN-1 endonuclease, DNA was prepared from lyophilized P. woesei bacteria (DSMZ 3773) as described (Zwickl et al., J. Bact., 172:4329 [1990]) with several changes. Briefly, one vial of P. woesei bacteria was rehydrated and resuspended in 0.5 ml of LB (Luria broth). The cells were centrifuged at 14,000 x g for 1 min and the cell pellet was resuspended in 0.45 ml of TE. Fifty microliters of 10% SDS was added and the mixture was incubated at RT for 5 min. The cell lysate was then extracted three time with 1:1 phenol:chloroform and three times with chloroform. Five hundred microliters of isopropanol was added to the extracted lysate and the DNA was pelleted by centrifugation at 14,000 x g for 10 min. The DNA pellet was washed in 0.5 ml of 70% ethanol and the DNA was pelleted again by centrifugation at 14,000 x g for 5 min.

The DNA pellet was dried and resuspended in 100 pl of TE and used for PCR reactions without further purification.

To generate a P. woesei FEN-1 gene fragment for cloning into an expression vector, low stringency PCR was attempted with primers complementary to the ends of the P. furiosus FEN-1 gene open reading frame. The sequences of the 5'-end and 3'-end primers are 5'-GATACCATGGGTGTCCCAATTGGTG-3 (SEQ ID NO:116) and 5'-TCGACGTCGACTTATCTCTTGAACCAACTTTCAAGGG (SEQ ID NO:117), 20 respectively. The high level of sequence similarity of protein homologs proteins other than FEN-1 proteins) from P. furiosus and P. woesei suggested that there was a high probability that the P. woesei FEN-1 gene could be amplified using primers containing sequences complementary to the P. furiosus FEN-1 gene. However, this approach was unsuccessful under several different PCR conditions.

25 The DNA sequence of FEN-1 genes from P. furiosus and M. jannaschii were aligned and blocks of sequence identity between the two genes were identified. These blocks were used to design internal primers complementary to sequences located internal to the and 3' ends of the ORF) for the FEN-1 gene that are complementary to the P. furiosus FEN-1 gene in those conserved regions. The sequences of the and 3'-internal primers are 5'-AGCGAGGGAGAGGCCCAAGC-3' (SEQ ID NO:118) and 5'-GCCTATGCCCTTTATTCCTCC-3' (SEQ ID NO:119), respectively. A PCR employing these internal primers was conducted using the AdvantageTM PCR kit and resulted in production of a major band of -300 bp.

184 Since the PCR with the internal primers was successful, reactions were attempted which contained mixtures of the internal (SEQ ID NOS:118 and 119) and external (SEQ ID NOS:116 and 117) primers. A reaction containing the 5'-end external primer (SEQ ID NO:116) and 3'-end internal primer (SEQ ID NO:119) resulted in the production of a 600 bp band and a reaction containing the 5'-end internal primer (SEQ ID NO:118) and 3'-end external primer (SEQ ID NO:117) resulted in the production of a 750 bp band. These overlapping DNA fragments were gel-purified and combined with the external primers (SEQ ID NOS:116 and 117) in a PCR reaction. This reaction generated a 1 kb DNA fragment containing the entire Pwo FEN-1 gene open reading frame. The resulting PCR product was gel-purified, digested, and ligated exactly as described above for the Mja FEN-1 gene PCR product. The resulting plasmid was termed pTrc99-PWFEN1. pTrc99-PWFENI was used to transform competent E. coli JM109 cells (Promega) using standard techniques.

d) Cloning and Expression Of A FEN-1 Endonuclease From Archaeoglobus fulgidus The preliminary Archaeoglobus fulgidus (Afu) chromosome sequence of 2.2 million bases was downloaded from the TIGR (The Institute for Genomic Research) world wide web site, and imported into a software program (MacDNAsis), used to analyze and manipulate DNA and protein sequences. The unannotated sequence was translated into all 6 of the °possible reading frames, each comprising approximately 726,000 amino acids. Each frame 20 was searched individually for the presence of the amino acid sequence "VFDG" (valine, Sphenylalanine, aspartic acid, glycine), a sequence which is conserved in the FEN-1 family.

The amino acid sequence was found in an open reading frame that contained other amino acid sequences conserved in the FEN-1 genes and which was approximately the same size as the other FEN-1 genes. The ORF DNA sequence is shown in SEQ ID NO:178, while the ORF protein sequence is shown in SEQ ID NO:179. Based on the position of this amino acid sequence within the reading frame, the DNA sequence encoding a putative FEN-1 gene was identified.

The sequence information was used to design oligonucleotide primers which were used for PCR amplification of the FEN-l-like sequence from A. fulgidus genomic DNA. Genomic DNA was prepared from A. fulgidus as described in Ex. 29a for M. janaschii, except that one vial (approximately 5 ml of culture) of live A. fulgidus bacteria from DSMZ (DSMZ #4304) was used. One microliter of the genomic DNA was used for PCR reaction as described in Ex. 29a. The 5' end primer is complementary to the 5' end of the Afu FEN-1 gene except it 185 has a 1 base pair substitution to create an Nco I site. The 3' end primer is complentary to the 3' end of the Afu FEN-1 gene downstream from the FEN-1 ORF except it contains a 2 base substitution to create a Sal I site. The sequences of the 5' and 3' end primers are 5'-CCGTCAACATTTACCATGGGTGCGGA-3' (SEQ ID NO:170) and 5'-CCGCCACCTCGTAGTCGACATCCTTTTCGTG (SEQ ID NO:171), respectively.

Cloning, expression and purification of the Afu FEN-1 gene was done as described in Examples 29a and 29d.

e) Cloning and Expression Of A FEN-1 Endonuclease From Methanobacterium thermoautotrophicum A tentative listing of all open reading frames of the Methanobacterium thermoautorrophicum (Mth) genome on the Genome Therapeutics world wide web page was searched for amino acid sequences conserved in the FEN-1 genes. The amino acid sequence "VFDG" (valine, phenylalanine, aspartic acid, glycine) was found in an open reading frame which also contained other conserved FEN-1 sequences. SEQ ID NO:181 provides the Mth FEN-1 ORF DNA sequence as indicated by Genome Therapeutics, while SEQ ID NO:182 provides the Mth FEN-1 ORF protein sequence as indicated by Genome Therapeutics.

However, this open reading frame was 259 amino acids in length, as compared to the other archael FEN-1 genes, which are approximately 325 amino acids long. To determine the cause of this discrepancy, the DNA sequence for Mth FEN-I was obtained in an identical manner as described above for Afu FEN-1.

Upon examination of the sequence, it was apparent that the open reading frame could be extended to 328 amino acids by deletion of a single base at about position 750 of the open reading frame. The additional amino sequence added by deleting one base is 39% identical to the same region of the P. furiosus FEN-1 gene. The DNA sequence of the putative Mth FEN-1 gene was used to design oligonucleotide primers complementary to the 5' and 3' ends of the gene. The 5' oligonucleotide is complementary to the 5' end of the Mth FEN-1 gene except that it contains 2 substitutions which create an NcoI site. The 3' oligonucleotide is complementary to the 3' end of the gene about 100 base pairs downstream of where it is believed that the true open reading frame ends. This region contains a natural PstI site. The 30 sequences of the 5' and 3' oligonucleotides are 5'-GGGTGTTCCCATGGGAGTTAAACTCAGG-3' (SEQ ID NO:172) and 5'-CTGAATTCTGCAGAAAAAGGGG-3' (SEQ ID NO:173), respectively.

o 186 Genomic DNA was prepared from 1 vial of frozen M thermoautotrophicum bacteria from ATCC (ATCC 29096) as described in Ex. 29a. PCR, cloning, expression, and purification of Mth FEN-I was done as described in Examples 29a and 29d. exccot Pstl was used instead of Sall. Sequencing of the cloned Mth FEN-1 gene revealed the presence of additional nucleotide when compared to the genome sequence published on the world wide web. This residue at position 775 of the FEN-I open reading frame causes a frame shift, creating the larger open reading frame that originally thought, based on comparison to the FEN genes from other organisms. SEQ ID NO: 182 provides the sequence of the Mth ORF DNA sequence of the present invention, while SEQ ID NO:183 provides the sequence of the Mth FEN-I protein sequence of the present invention.

f) Large Scale Preparation of Recombinant Thermostable FEN-1 Proteins The Mja, Pwo and Pfu FEN-1 proteins were purified by the following technique which is derived from a Taq DNA polymerase preparation protocol (Engelke et al., Anal. Biochem., 191:396 [1990]) as follows. E. coli cells (strain JM109) containing either pTrc99-PFFEN1, pTrc99-PWFEN1, or pTrc99-MJFEN1 were inoculated into 3 ml of LB (Luria Broth) containing 100 pg/ml ampicillin and grown for 16 hrs at 37 0 C. The entire overnight culture was inoculated into 200 ml or 350 ml of LB containing 100 pg/ml ampicillin and grown at 37 0 C with vigorous shaking to an Ao0 of 0.8. IPTG (1 M stock solution) was added to a final concentration of 1 mM and growth was continued for 16 hrs at 37 0

C.

The induced cells were pelleted and the cell pellet was weighed. An equal volume of 2X DG buffer (100 mM Tris-HC1, pH 7.6, 0.1 mM EDTA) was added and the pellet was resuspended by agitation. Fifty mg/ml lysozyme (Sigma) were added to 1 mg/ml final concentration and the cells were incubated at room temperature for 15 min. Deoxycholic acid (10% solution) was added dropwise to a final concentration of 0.2 while vortexing. One volume of H 2 0 and 1 volume of 2X DG buffer was added and the resulting mixture was sonicated for 2 minutes on ice to reduce the viscosity of the mixture. After sonication, 3 M

(NH

4 2 SO, was added to a final concentration of 0.2 M and the lysate was centrifuged at 14000 x g for 20 min at 4°C. The supernatant was removed and incubated at 70 0 C for min at which time 10% polyethylimine (PEI) was added to 0.25%. After incubation on ice for 30 min., the mixture was centrifuged at 14,000 x g for 20 min at 4°C. At this point, the *o supernatant was removed and the FEN-1 proteins was precipitated by the addition of (NH,)2SO4 as follows.

187- For the Pwo and the Pfu FEN-1 preparations, the FEN-I protein was precipitated by the addition of 2 volumes of 3 M (NH 4 2 SO,. The mixture was incubated overnight at room temperature for 16 hrs and the protein was centrifuged at 14,000 x g for 20 min at 4 0 C. The protein pellet was resuspended in 0.5 ml of Q buffer (50 mM Tris-HC, pH 8.0, 0.1 mM EDTA, 0.1% Tween 20). For the Mja FEN-I preparation, solid (NH 4 2 SO, was added to a final concentration of 3 M saturated), the mixture was incubated on ice for 30 min, and the protein was spun down and resuspended as described above.

The resuspended protein preparations were quantitated by determination of the A 9 and aliquots containing 2-4 pg of total protein were electrophoresed on a 10 SDS polyacrylamide gel (29:1 acrylamide: bis-acrylamide) and stained with Coomassie Brilliant Blue R; the results are shown in Fig. In Fig. 75, lane 1 contains molecular weight markers (Mid-Range Protein Molecular Weight Markers; Promega); the size of the marker proteins is indicated to the left of the gel.

Lane 2 contains purified Cleavase® BN nuclease: lanes 3-5 contain extracts prepared from E.

coli expressing the Pfu, Pwo and Mja FEN-1 nucleases, respectively. The calculated using a translation of the DNA sequence encoding the nuclease) molecular weight of the Pfu FEN-1 nuclease is 38,714 daltons and the calculated molecular weight for the Mja FEN-1 nuclease is 37.503 Daltons. The Pwo and Pfu FEN-1 proteins co-migrated on the SDS-PAGE gel and therefore, the molecular weight of the Pwo FEN-1 nuclease was estimated to be 38.7 20 kDa.

e-g) Activity Assays Using FEN-1 Endonucleases i) Mixed Hairpin Assay The Cleavase® BN nuclease has an approximately 60-fold greater affinity for a 12 base pair stem-loop structure than an 8 base pair stem-loop DNA structure. As a test for activity differences between the Cleavase® BN nuclease and the FEN-1 nucleases, a mixture of oligonucleotides having either a 8 or a 12 bp stem-loop (see Fig. 71 which depicts the S-33 and 11-8-0 oligonucleotides) was incubated with an extract prepared from E. coli cells overexpressing the Mja FEN-1 nuclease (prepared as described above). Reactions contained 0.05 pM of oligonucleotides S-33 (SEQ ID NO:120) and 11-8-0 (SEQ ID NO:121) (both oligonucleotides contained 5'-fluorescein labels), 10 mM MOPS, pH 7.5, 0.05% 0.05% NP-40, 1 mM MnCI,. Reactions were heated to 90 0 C for 10 seconds, cooled to 55 0

C,

then 1 pl of crude extract (Mja FEN-1) or purified enzyme (Cleavase® BN nuclease) was added and the mixtures were incubated at 55°C for 10 minutes; a no enzyme control was also 188

I

run. The reactions were stopped by the addition of formamide/EDTA, the samples were electrophoresed on a denaturing 20% acrylamide gel and visualized on a Hitachi FMBIO 100 flir-rescence imager. The resulting image is shown in Fig. 76.

In Fig. 76, lane 1 contains the reaction products generated by the Cleavase® BN nuclease, lane 2 contains the reaction products from the no enzyme control reaction and lane 3 contains the reaction products generated by the Mja FEN-I nuclease. The data shown in Fig. 76 demonstrates that the Cleavase® BN nuclease strongly prefers the S33 structure (12 bp stem-loop) while the Mja FEN-1 nuclease cleaves structures having either an 8 or a 12 bp stem-loop with approximately the same efficiency. This shows that the Mja FEN-1 nuclease has a different substrate specificity than the Cleavase® BN nuclease, a useful feature for InvaderTM assays or CFLP® analysis as discussed in the Description of the Invention.

EXAMPLE Terminal Deoxynucleotidyl Transferase Selectively Extends The Products Of InvaderM-Directed Cleavage The majority of thermal degradation products of DNA probes will have a phosphate at the 3'-end. To investigate if the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) can tail or polymerize the aforementioned 3'-end phosphates add nucleotide triphosphates to the 3' end) the following experiment was performed.

To create a sample containing a large percentage of thermal degradation products, the fluorescein-labeled oligonucleotide 34-078-01 (SEQ ID NO:73) (200 pmole) was incubated in 100 il 10 mM NaCO 3 (pH 10.6), 50 mM NaCI at 95 0 C for 13 hours. To prevent evaporation, the reaction mixture was overlaid with 60 pl ChillOutTMl4 liquid wax (MJ Research). The reaction mixture was then divided into two equal aliquots (A and Aliquot A was mixed with one-tenth volume 3M NaOAc followed by three volumes ethanol and stored at -20 0 C. Aliquot B was dephosphorylated by the addition of 0.5 pl of IM MgCl 2 and 1 pl of lunit/pl Calf Intestine Alkaline Phosphatase (CIAP) (Promega), with incubation at 37 0 C for 30 minutes. An equal volume of phenol:chloroform: isoamyl alcohol (24:24:1) was added to the sample followed by vortexing for one minute and then centrifugation 5 minutes at maximum speed in a microcentrifuge to separate the phases. The aqueous phase was removed to a new tube to which one-tenth volume 3M NaOAc, and three volumes ethanol was added followed by storage at -20 0 C for 30 minutes. Both aliquots (A and B) were then 189centrifuged for 10 minutes at maximum speed in a microcentrifuge to pellet the DNA. The pellets were then washed two times each with 80% ethanol and then desiccated to dryness.

The dried pellets were then dissolved in 70 pl ddH,O each.

The TdT reactions were conducted as follows. Six mixes were assembled, all mixes contained 10 mM TrisOAc (pH 10 mM MgOAc, 50 mM KC1, and 2 mM dATP. Mixes I and 2 contained one pmole of untreated 34-078-01 (SEQ ID NO:73), mixes 3 and 4 contained 2 pl of aliquot A (above), mixes 5 and 6 contained 2 .1 of aliquot B (above). To each 9 pl of mixes 1,3 and 5, 1 p1 ddHO was added, to each 9 p1 of mixes 2, 4, and 6, 1 pl of 20 units/pl TdT (Promega) was added. The mixes were incubated at 37 0 C for 1 hour and then the reaction was terminated by the addition of 5 p1 95% formamide with 10 mM EDTA and 0.05% marker dyes. Five microliters of each mixture was resolved by electrophoresis through a 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate (pH 1.4 mM EDTA, and imaged as described in Ex. 21; a 505 nm filter was used with the FMBIO Image Analyzer. The resulting imager scan is shown in Fig. 81.

In Fig. 81, lanes 1, 3 and 5 contain untreated 34-078-01 (SEQ ID NO:73), heat-degraded 34-078-01 (SEQ ID NO:73), and heat-degraded, dephosphorylated, 34-078-01 (SEQ ID NO:73), respectively incubated in the absence of TdT. Lanes 2, 4 and 6 contain, untreated 34-078-01 (SEQ ID NO:73), heat-degraded 34-078-01 (SEQ ID NO:73), and 20 heat-degraded, dephosphorylated, 34-078-01 (SEQ ID NO:73), respectively incubated in the presence of TdT.

As shown in Fig. 81, lane 4, TdT was unable to extend thermal degradation products which contain a 3'-end phosphate group, and selectively extends molecules which have a 3'end hydroxyl group.

9 o• 190 EXAMPLE 31 Specific TdT Tailing Of The Products Of Invader"'-Directed Cleavage With Subsequent Capture And Detection On Nitrocellulose Supports When TdT is used to extend the specific products of cleavage, one means of detecting the tailed products is to selectively capture the extension products on a solid support before visualization. This example demonstrates that the cleavage products can be selectively tailed by the use of TdT and deoxynucleotide triphosphates, and that the tailed products can be visualized by capture using a complementary oligonucleotide bound to a nitrocellulose support.

To extend the cleavage product produced in an InvaderTM-directed cleavage reaction, the following experiment was performed. Three reaction mixtures were assembled, each in a buffer of 10 mM MES (pH 0.5%Tween-20, 0.5% NP-40. The first mixture contained fmols of target DNA-M13mpl8, 10 pmols of probe oligonucleotide 32-161-2 (SEQ ID NO:71; this probe oligonucleotide contains 3' ddC and a Cy3 amidite group near the 3' end), and 5 pmols of Invader" oligonucleotide 32 161-1 (SEQ ID NO:70; this oligonucleotide contains a 3' ddC). The second mixture contained the probe and Invader" oligonucleotides without target DNA. The third mixture was the same as the first mixture, and contained the same probe sequence, but with a 5' fluorescein label [oligonucleotide 32-161-4 (SEQ ID NO:72; this oligonucleotide contains a 3' ddC, 5' fluorescein label, and a Cy3 amidite group 20 near the 3' end)], so that the InvaderTM-directed cleavage products could be detected before and after cleavage by fluorescence imaging. The probe only control sample contained pmols of oligonucleotide 32-161-2 (SEQ ID NO:71). Each 3 [il of enzyme mix contained ng of Cleavase® DN nuclease in 7.5 mM MgCl,. The TdT mixture (per each 4 p1l) S contained: 10U of TdT (Promega), 1 mM CoCI 2 50 mM KC1, and 100 M of dTTP. The Invader" cleavage reaction mixtures described above were assembled in thin wall tubes, and the reactions were initiated by the addition of 3 pl of Cleavase® DN enzyme mix. The reactions were incubated at 65 0 C for 20 min. After cooling to 37 0 C, 4 ll of the TdT mix was added and the samples were incubated for 4 min at 37 0 C, Biotin-16-dUTP was then added to 100 pM and the samples were incubated for 50 min at 37 0 C. The reactions were "30 terminated by the addition of 1 pl of 0.5 M EDTA.

To test the efficiency of tailing the products were run on an acrylamide gel. Four Smicroliters of each reaction mixture was mixed with 2.6 pl of 95% formamide, 10 mM EDTA and 0.05% methyl violet and heated to 90 0 C for 1 min, and 3 pl were loaded on a 191 7, denaturing acrylamide gel (19:1 cross-linked with 7 M urea, in buffer containing mM Tris-Borate (pH 1.4 mM EDTA. A marker [DX174-Hinfl (fluorescein labeled)] also was loaded. After electrophoresis, the gel was analyzed using a FMBIO-100 Image Analyzer (Hitachi) equipped with a 505 nm filter. The resulting scan is shown in Fig. 82.

In Fig. 82, lane 1 contained the probe 32-161-2 only, without any treatment. Lanes 2 and 3 contained the products of reactions run without target DNA, without or with subsequent TdT tailing, respectively. Lanes 4 and 5 contained the products of reactions run with target DNA, probe oligonucleotide 32-161-2 (SEQ ID NO:71) and InvaderTM oligonucleotide 32- 161-1 (SEQ ID NO:70), without or with subsequent TdT tailing, respectively. Lanes 6 and 7 show the products of reactions containing target DNA, probe oligonucleotide 32-161-4 (SEQ ID NO:72) and InvaderT" oligonucleotide 32-161-1 (SEQ ID NO:70), without or with subsequent TdT tailing, respectively. Lane M contains the marker >X174-Hinfl.

The reaction products in lanes 4 and 5 are the same as those seen in lanes 6 and 7, except that the absence of a 5' fluorescein on the probe prevents detection of the released product (indicated as near the bottom of the gel) or the TdT extended 5' product (indicated as near the top of the gel). The Cy3-labeled 3' portion of the cleaved probe is visible in all of these reactions (indicated as just below the center of the gel).

To demonstrate detection of target-dependent Invader-directed cleavage products on a solid support, the reactions from lanes 3 and 5 were tested on the Universal GeneCombO® 20 (Bio-Rad) which is a standard nitrocellulose matrix on a rigid nylon backing styled in a comb format, as depicted in Fig. 79. Following the manufacturer's protocol, with one modification: 10 p of the Invader-directed cleavage reactions were used instead the recommended 10% of a PCR. To capture the cleavage products, 2.5 pmols of the capture oligonucleotide 59-28-1 (SEQ ID NO:133) were spotted on each tooth. The capture and visualization steps were conducted according to the manufacturer's directions. The results are shown in Fig. 79.

In Fig. 79, teeth numbered 6 and 7 show the capture results of reactions performed without and with target DNA present. Tooth 8 shows the kit positive control.

The darkness of the spot seen on tooth 7, when compared to tooth 6, clearly indicates that products of InvaderTM-directed cleavage assays may be specifically detected on solid supports. While the Universal GeneComb)® was used to demonstrate solid support capture in this instance, other support capture methods known to those skilled in the art would be *o equally suitable. For example, beads or the surfaces of reaction vessels may easily be coated with capture oligonucleotides so that they can then be used in this step. Alternatively, similar 192 solid supports may easily be coated with streptavidin or antibodies for the capture of biotinor hapten-tagged products of the cleavage/tailing reaction. In any of these embodiments, the products may be appropriately visualized by detecting the resulting fluorescence, chemiluminescence, colorimetric changes, radioactive emissions, optical density change or any other distinguishable feature of the product.

EXAMPLE 32 Comparison Of The Effects Of Invasion Length and 5' Label Of The Probe On InvaderTM-Directed Cleavage By The Cleavase® A/G and Pfu FEN-1 Nucleases To investigate the effect of the length of invasion as well as the effect of the type of dye on ability of Pfu FEN-1 and the Cleavase® A/G nuclease to cleave 5' arms, the following experiment was performed. Three probes of similar sequences labeled with either fluorescein. TET, or Cy3, were assembled in reactions with three InvaderTM oligonucleotides which created overlapping target hybridization regions of eight, five, and three bases along the target nucleic acid, M13mpl8.

The reactions were conducted as follows. All conditions were performed in duplicate.

Enzyme mixes for Pfu FEN-1 and the Cleavase® A/G nuclease were assembled. Each 2 Pl of the Pfu FEN-I mix contained 100 ng of Pfu FEN-1 (prepared as described in Ex. 28) and mM MgCl,. Each 2 pl of the Cleavase® A/G mix contained 5.3 ng of the Cleavase® 20 A/G nuclease and 4.0 mM MnCI 2 Six master mixes containing buffer, M13mpl8, and InvaderTM oligonucleotides were assembled. Each 7 .il of mixes 1-3 contained 1 fmol M13mpl8,d 10 pmoles InvaderTM oligonucleotide (34-078-4 [SEQ ID NO:50], 24-181-2 [SEQ ID NO:76], or 24-181-1 [SEQ ID NO:75] in 10 mM MOPS (pH 150 mM LiCI. Each 7 pl of mixes 4-6 contained 1 fmol of M13mpl8, 10 pmoles of InvaderTM oligonucleotide (34-078-4 [SEQ ID NO:50], 24-181-2 [SEQ ID NO:76], or 24-181-1 [SEQ ID NO:75]) in mM Tris (pH Mixtures 1-6 were then divided into three mixtures each, to which was added either the fluorescein-labeled probe (oligonucleotide 34-078-01; SEQ ID NO:73), the Cy3-labeled probe (oligonucleotide 43-20; SEQ ID NO:74) or the TET-labeled probe (oligonucleotide 90; SEQ ID NO:43 containing a 5' TET label). Each 7 pl of all mixtures contained 10 pmoles of corresponding probe. The DNA solutions described above were covered with 10 pl of ChillOut® evaporation barrier (MJ Research) and brought to 68 0

C.

193 The reactions made from mixes 1-3 were started with 2 Vl of the Cleavase® A/G nuclease mix, and the reactions made from mixes 4-6 were started with 2 p~ of the Pfu FEN- 1 mix. After 30 minutes at 68 0 C, the reactions were terminated by the addition of 8 Cl1 of formamide with 10 mM EDTA and 0.05% marker dyes. Samples were heated to for 1 minute immediately before electrophoresis through a 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate (pH 1.4 mM EDTA. The products of the cleavage reactions were visualized following electrophoresis by the use of a Hitachi FMBIO fluorescence imager. Results from the fluorescein-labeled probe are shown in Fig. 80, results from the Cy3-labeled probe in Fig. 81, and results from the TET-labeled probe in Fig. 82. In each of these figures the products of cleavage by Cleavase® A/G are shown in lanes 1-6 and the products of cleavage by Pfu FEN- are shown in lanes 7-12. In each in case the uncut material appears as a very dark band near the top of the gel, indicated by a on the left. The products of cleavage directed by Invader oligonucleotides with 8, 5 or 3 bases of overlap the region was 8, 5, or 3 nt long) are shown in the first, second and third pair of lanes in each set, respectively and the released labeled 5' ends from these reactions are indicated by the numbers 8, 5, and 3 on the left.

Note that in the cleavage reactions shown in Fig. 81 the presence of the positively charged Cy3 dye causes the shorter products to migrate more slowly than the larger products. These products do not contain any additional positive charges, amino modifications as used in 20 Ex. 23, and thus still carry a net negative charge, and migrate towards the positive electrode in a standard electrophoresis run.

i. It can be seen from these data that the Cleavase® A/G and Pfu FEN-1 structurespecific nucleases respond differently to both dye identity and to the size of the piece to be cleaved from the probe. The Pfu FEN-I nuclease showed much less variability in response to dye identity than did the Cleavase® A/G nuclease, showing that any dye would be suitable for use with this enzyme. In contrast, the amount of cleavage catalyzed by the Cleavase® A/G nuclease varied substantially with dye identity. Use of the fluorescein dye gave results very close to those seen with the Pfu FEN-1 nuclease, while the use of either Cy3 or TET gave dramatically reduced signal when compared to the Pfu FEN-l reactions. The one exception to this was in the cleavage of the 3 nt product carrying a TET dye (lanes 5 and 6, Fig. 82), in which the Cleavase® A/G nuclease gave cleavage at the same rate as the Pfu FEN-I nuclease.

These data indicate that, while Cleavase® A/G may be used to cleave probes labeled with o* 194 these other dyes, the Pfu FEN-I nuclease is a preferred nuclease for cleavage of Cy3- and TET-labeled probes.

EXAMPLE 33 Examination Of The Effects Of A 5' Positive Charge On The Rate Of Invasive Cleavage Using The Cleavase® A/G Or Pfu FEN-1 Nucleases To investigate whether the positive charges on 5' end of probe oligonucleotides containing a positively charged adduct(s) charge reversal technology or CRT probes as described in Exs. 23 and 24) have an effect on the ability of the Cleavase® A/G or Pfu FEN- 1 nucleases to cleave the 5' arm of the probe, the following experiment was performed.

Two probe oligonucleotides having the following sequences were utilized in InvaderTM reactions: Probe 34-180-1: (N-Cy3)TNHTNH 2

CCAGAGCCTAATTTGCC

AGT(N-fluorescein)A, where N represents a spacer containing either the Cy3 or fluorescein group (SEQ ID NO:77) and Probe 34-180-2: CCTAATTTGCCAGT-(N-fluorescein)A, where N represents a spacer containing either the TET or fluorescein group (SEQ ID NO:78). Probe 34-180-1 has amino-modifiers on the two end T residues and a Cy3 label on the 5' end, creating extra positive charges on the 5' end.

Probe 34-180-2 has a TET label on the 5' end, with no extra positive charges. The fluorescein label on the 3' end of probe 34-180-1 enables the visualization of the 3' cleaved 20 products and uncleaved probes together on an acrylamide gel run in the standard direction with the DNA migrating toward the positive electrode). The 5' cleaved product of probe 34-180-1 has a net positive charge and will not migrate in the same direction as the uncleaved probe, and is thus visualized by resolution on a gel run in the opposite direction with this DNA migrating toward the negative electrode).

The cleavage reactions were conducted as follows. All conditions were performed in duplicate. Enzyme mixes for the Pfu FEN-1 and Cleavase® A/G nucleases were assembled.

Each 2 il of the Pfu FEN-1 mix contained 100 ng of Pfu FEN-1 (prepared as described in Ex. 28) and 7.5 mM MgCl. Each 2 pl of the Cleavase® A/G nuclease mix contained 26.5 ng of Cleavase® A/G nuclease and 4.0 mM MnCI 2 Four master mixes containing buffer, M13mpl8, and InvaderTM oligonucleotides were assembled. Each 7 p1 of mix 1 contained :"*fmol M13mpl8, 10 pmoles InvaderTM oligonucleotide 123 (SEQ ID NO:79) in 10 mM HEPES (pH Each 7 l1 of mix 2 contained 1 fmol M13mpl8, 10 pmoles InvaderTM oligonucleotide 123 in 10 mM HEPES (pH Each 7 pl of mix 3 contained 5 fmol 195 M13mpl8, 10 pmoles InvaderTM oligonucleotide 123 in 10 mM HEPES (pH 250 mM KGlu. Each 7 pl of mix 4 contained 1 fmol Ml3mpl8, 10 pmoles InvaderTM oligonucleotide 123 in 10 mM HEPES (pH 250 mM KGlu. For every 7 pl of each mix. 10 pmoles of either probe 34-180-1 (SEQ ID NO:77) or probe 34-180-2 (SEQ IDNO:78) was added. The DNA solutions described above were covered with 10 pil of ChillOut® evaporation barrier (MJ Research) and brought to 65 0 C. The reactions made from mixes 1-2 were started by the addition of 2 pl of the Pfu FEN-1 mix, and the reactions made from mixes 3-4 were started by the addition of 2 pl of the Cleavase® A/G nuclease mix. After 30 minutes at 65 0 C, the reactions were terminated by the addition of 8 pl of 95% formamide containing 10 mM EDTA. Samples were heated to 90 0 C for 1 minute immediately before electrophoresis through a 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate (pH 1.4 mM EDTA and a 20% native acrylamide gel (29:1 cross-linked) in a buffer containing 45 mM Tris-Borate (pH 1.4 mM EDTA.

The products of the cleavage reactions were visualized following electrophoresis by the use of a Hitachi FMBIO fluorescence imager. The resulting images are shown in Figs. 83.

Fig. 83A shows the denaturing gel which was run in the standard electrophoresis direction, and Fig. 83B shows the native gel which was run in the reverse direction. The reaction products produced by Pfi FEN-1 and Cleavase® A/G nucleases are shown in lanes 1-8 and 9-16, respectively. The products from the 5 fmol M13mpl8 and 1 fmol Ml3mpl8 reactions 20 are shown in lanes 1-4, 9-12 (5 fmol) and 5-8, 13-16 (1 fmol). Probe 34-180-1 is in lanes 1-2, 5-6. 9-10, 13-14 and probe 34-180-2 is in lanes 3-4, 7-8, 11-12, 15-16.

The fluorescein-labeled 3' end fragments from all cleavage reactions are shown in Fig.

83A, indicated by a mark at the left. The 3 nt 5' TET-labeled products are not visible in this figure, while the 5' Cy3-labeled products are shown in Fig. 83B.

The 3' end bands in Fig. 83A can be used to compare the rates of cleavage by the different enzymes in the presence of the different 5' end labels. It can be seen from this band that regardless of the amount of target nucleic acid present, both the Pfu FEN-1 and the Cleavase® A/G nucleases show more product from the 5' TET-labeled probe. With the Pfu FEN-I nuclease this preference is modest, with only an approximately 25 to 40% increase in signal. In the case of the Cleavase® A/G nuclease, however, there is a strong preference for the 5' TET label. Therefore, although when the charge reversal method is used to resolve the products, a substantial amount of product is observed from the Cleavase® A/G nuclease- 196catalyzed reactions, the Pfu FEN-I nuclease is a preferred enzyme for cleavage of Cy3labeled probes.

EXAMPLE 33 The Use Of Universal Bases In The Detection Of Mismatches By InvaderTM-Directed Cleavage The term "degenerate base" refers to a base on a nucleotide that does not hydrogen bond in a standard "Watson-Crick" fashion to a specific base complement, A to T and G to C. For example, the inosine base can be made to pair via one or two hydrogen bonds to all of the natural bases (the "wobble" effect) and thus is called degenerate. Alternatively, a degenerate base may not pair at all; this type of base has been referred to as a "universal" base because it can be placed opposite any nucleotide in a duplex and, while it cannot contribute stability by base-pairing, it does not actively destabilize by crowding the opposite base. Duplexes using these universal bases are stabilized by stacking interactions only. Two examples of universal bases, 3-nitropyrrole and 5-nitroindole, are shown in Fig. 84. In hybridization, placement of a 3-nitropyrrole three bases from a mismatch position enhances the differential recognition of one base mismatches. The enhanced discrimination seems to come from the destabilizing effect of the unnatural base an altered T, in close proximity to the mismatch). To test this same principle as a way of sensitively detecting mismatches 20 using the InvaderTM-directed cleavage assay, InvaderTM oligonucleotides were designed using •the universal bases shown in Fig. 84, in the presence or absence of a natural mismatch. In these experiments, the use of single nitropyrrole bases or pairs of nitroindole bases that flank S*the site of the mismatch were examined.

The target, probe and Invader T M oligonucleotides used in these assays are shown in Fig. 85. A 43 nucleotide oligonucleotide (oligonucleotide 109; SEQ ID NO:83) was used as the target. The probe oligonucleotide (oligonucleotide 61; SEQ ID NO:61) releases a net positively charged labeled product upon cleavage. In Fig. 85, the InvaderTM oligonucleotide is shown schematically above the target oligonucleotide as an arrow; the large arrowhead indicates the location of the mismatch between the InvaderTM oligonucleotides and the target.

Under the target oligonucleotide, the completely complementary, all natural no universal bases) Invader TM oligonucleotide (oligonucleotide 67; SEQ ID NO:62) and a composite of Invader T M oligonucleotides containing universal bases on either side of the mismatch are shown. The following InvaderTM oligonucleotides were employed: oligonucleotide 197- 114 (SEQ ID NO:86) which contains a single nt mismatch; oligonucleotide 115 (SEQ ID NO:87) which contains two 5-nitroindole bases and no mismatch; oligonucleotide 116 (SEQ ID NO:88) which contains two 5-nitroindole bases and a single nt mismatch; oligonucleotide 112 (SEQ ID NO:84) which contains one 3-nitropyrrole base and no mismatch; oligonucleotide 113 (SEQ ID NO:85) which contains one 5-nitropyrrole base and a single nt mismatch; and oligonucleotide 67 (SEQ ID NO:62) which is completely complementary to the target.

The InvaderTM-directed cleavage reactions were carried out in 10 p1 of 10 mM MOPS (pH 100 mM KC1, containing 1 pM of the appropriate invading oligonucleotide (oligonucleotides 67, 112-116), 10 nM synthetic target 109, 1 .M Cy-3 labeled probe 61 and 2 units of Cleavase® DV (prepared as described in Ex. 27). The reactions were overlaid with Chill-Out® liquid wax. brought to the appropriate reaction temperature, 52 0 C, 55 0 C, or 58°C and initiated with the addition of 1 pil of 40 mM MnCI 2 Reactions were allowed to proceed for I hour and were stopped by the addition of 10 pl formamide. One fourth of the total volume of each reaction was loaded onto 20% non-denaturing polyacrylamide gels which were electrophoresed in the reverse direction. The products were visualized using an Hitachi FMBIO-100 fluorescent scanner using a 585 nm filter. The resulting images are shown in Figs. 86A-C. In each panel, lanes 1-6 contain reactions products from reactions using InvaderTM oligonucleotide 67, 114, 115, 116, 112 and 113, respectively. Reactions run at 20 52 0 C, 55°C and 58 0 C are shown in Panels A, B and C, respectively.

These data show that two flanking 5-nitroindoles display a significantly greater differentiation then does the one 3-nitropyrrole system, or the all natural base hybridization, and this increased sensitivity is not temperature dependent. This demonstrates that the use of universal bases is a useful means of sensitively detecting single base mismatches between the target nucleic acid and the complex of detection oligonucleotides of the present invention.

EXAMPLE Detection Of Point Mutations in The Human Ras Oncogene Using A Miniprobe It is demonstrated herein that very short probes can be used for sensitive detection of target nucleic acid sequences (Ex. 37). In this example, it is demonstrated that the short Sprobes work very poorly when mismatched to the target, and thus can be used to distinguish a given nucleic acid sequence from a close relative with only a single base difference. To test this system synthetic human ras oncogene target sequences were created that varied from each 198 other at one position. Oligonucleotide 166 (SEQ ID NO:93) provided the wild-type ras target sequence. Oligonucleotide 165 (SEQ ID NO:92) provided the mutant ras target sequence.

The sequence of theCe !ironcni!eitide are honwn in Fig 8 7 and the site of the seauence variation in the site corresponding to codon 13 of the ras gene is indicated. The InvaderT" oligonucleotide (oligonucleotide 162) has the sequence: TTGCCTACGA-3' (SEQ ID NO:90), where the indicates thiol linkages [i.e.,_these are 2'- The miniprobe (oligonucleotide 161) has the sequence: 5'-(N-Cy3) TN,,T,4,CACCAG-3' (SEQ ID NO:89) and is designed to detect the mutant ras target sequence it is completely complementary to oligonucleotide 165). The stacker oligonucleotide (oligonucleotide 164) has the sequence: AsCsTsAsCCAC AAGTTTATATTCAG-3' (SEQ ID NO:91). A schematic showing the assembly of these oligonucleotides into a cleavage structure is depicted in Fig. 87.

Each cleavage reaction contained 100 nM of both the invading (oligonucleotide 162) and stacking (oligonucleotide 164) oligonucleotides, 10 pM Cy3-labeled probe (oligonucleotide 161) and 100 pM of either oligonucleotide 165 or oligonucleotide 166 (target DNA) in 10 pl of 10 mM HEPES (pH 250 mM KGlu, 4 mM MnCI,. The DNA mixtures were overlaid with mineral oil, heated to 90 0 C for 15 sec then brought to a reaction temperature of 470, 500, 53° or 56 0 C. Reactions were initiated by the addition of 1 pl of 100 ng/pl Pfu FEN-1. Reactions were allowed to proceed for 3 hours and stopped by the addition 20 of 10 pl formamide. One fourth of the total volume of each reaction was loaded onto a non-denaturing polyacrylamide gel which was electrophoresed in the reverse direction. The gel was scanned using an Hitachi FMBIO-100 fluorescent scanner fitted with a 585 nm filter.

*.and the resulting image is shown in Fig. 88.

In Fig. 88, for each reaction temperature tested, the products from reactions containing either the mutant ras target sequence (oligonucleotide 165) or the wild-type (oligonucleotide 166) are shown.

These data demonstrate that the miniprobe can be used to sensitively discriminate between sequences that differ by a single nucleotide. The miniprobe was cleaved to produce a strong signal in the presence of the mutant target sequence, but little or no miniprobe was cleaved in the presence of the wild-type target sequence. Furthermore, the discrimination between closely related targets is effective over a temperature range of at least 10 0 C, which is a much broader range of temperature than can usually be tolerated when the selection is based on hybridization alone hybridization with ASOs). This suggests that the enzyme may be 199a factor in the discrimination, with the perfectly matched miniprobe being the preferred substrate when compared to the mismatched miniprobe. Thus, this system provides sensitive and specific detection of target nucleic acid sequences.

EXAMPLE 36 Effects of 3' End Identity On Site Of Cleavage Of A Model Oligonucleotide Structure As described in the examples above, structure-specific nucleases cleave near the junction between single-stranded and base-paired regions in a bifurcated duplex, usually about one base pair into the base-paired region. It was shown in Ex. 11 that thermostable nucleases, including those of the present invention Cleavase® BN nuclease, Cleavase® A/G nuclease), have the ability to cleave a greater distance into the base paired region when provided with an upstream oligonucleotide bearing a 3' region that is homologous to a region of the subject duplex, as shown in Fig. 30. It has also been determined that the 3' terminal nucleotide of the invader oligonucleotide may be unpaired to the target nucleic acid.

and still shift cleavage the same distance into the down stream duplex as when paired. It is shown in this example that it is the base component of the nucleotide, not the sugar or phosphate, that that is necessary to shift cleavage.

Figs. 89A and B shows a synthetic oligonucleotide which was designed to fold upon itself which consists of the following sequence: 20 GGTCGCTGTCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3' (SEQ ID NO:40). This oligonucleotide is referred to as the "S-60 Hairpin." The 15 basepair hairpin formed by this oligonucleotide is further stabilized by a "tri-loop" sequence in the loop end three nucleotides form the loop portion of the hairpin) (Hiraro et al., Nucleic Acids Res.

22(4): 576 [1949]). Fig. 89B shows the sequence of the P-15 oligonucleotide (SEQ ID NO:41) and the location of the region of complementarity shared by the P-15 and hairpin oligonucleotides. In addition to the P-15 oligonucleotide shown, cleavage was also tested in the presence of the P-14 oligonucleotide (SEQ ID NO:122) (P-14 is one base shorter on the 3' end as compared to P-15), the P-14 with an abasic sugar (P-14d; SEQ ID and the P14 with an abasic sugar with a 3' phosphate (P-14dp; SEQ ID NO:81). A oligonucleotide with a 3' phosphate, P-15p (SEQ ID NO:82) was also examined. The black arrows shown in Fig. 89 indicate the sites of cleavage of the S-60 hairpin in the absence (top structure; A) or presence (bottom structure; B) of the P-15 oligonucleotide.

200- The S-60 hairpin molecule was labeled on its 5' end with fluorescein for subsequent detection. The S-60 hairpin was incubated in the presence of a thermostable 5' nuclease in the presence or the absence of the P-15 oligonucleotide. The presence of the full duplex which can be formed by the S-60 hairpin is demonstrated by cleavage with the Cleavase® BN 5' nuclease. in a primer-independent fashion in the absence of the P-15 oligonucleotide).

The release of 18 and 19-nucleotide fragments from the 5' end of the S-60 hairpin molecule showed that the cleavage occurred near the junction between the single and double stranded regions when nothing is hybridized to the 3' arm of the S-60 hairpin (Fig. 31, lane 2).

The reactions shown in Fig. 89C were conducted in 10 pl IX CFLP buffer with 1 mM MnCI, and 50 mM K-Glutamate, in the presence of 0.02 pM S-60, 0.5 4M InvaderTM oligonucleotide and 0.01 ng per pil Cleavase® BN nuclease. Reactions were incubated at 0 C for 5 minutes and stopped by the addition of 8 pl of stop buffer (95% formamide. mM EDTA. 0.02% methyl violet). Samples were heated to 75°C for 2 min immediately before electrophoresis through a 15% acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Gels were then analyzed with a FMBIO-100 Image Analyzer (Hitachi) equipped with 505 nm filter. The resulting image is shown in Fig. 89C.

In Fig. 89C lane 1 contains products from the no enzyme control; lane 2 contains products from a reaction run in the absence of an InvaderTM oligonucleotide; lanes 3-6 contain 20 products from reactions run the presence of the P-14d, P-14dp, P-15 and P-15p Invader

T

oligonucleotides, respectively.

From the data shown in Fig. 89C. it can be seen that the use of the P-15 Invader T M oligonucleotide produces a shift in the cleavage site, while the P14 InvaderTM oligonucleotide with either a ribose (P14d) or a phosphorylated ribose (Pl4dp) did not This indicates that the 15th residue of the Invader T M oligonucleotide must have the base group attached to promote *9 the shift in cleavage. Interestingly, the addition of phosphate to the 3' end of the oligonucleotide apparently reversed the shifting of cleavage site. The cleavage in this lane may in fact be cleavage in the absence of an Invader

T

m oligonucleotide as is seen in lane 2.

In experiments with 5' dye-labeled InvaderTM oligonucleotides with 3' phosphate groups these oligonucleotides have been severely retarded in gel migration, suggesting that either the enzyme or another constituent of the reaction BSA) is able to bind the 3' phosphate irrespective of the rest of the cleavage structure. If the InvaderTM oligonucleotides are indeed -201 being sequestered away from the cleavage structure, the resulting cleavage of the S-60 hairpin would occur in a "primer-independent' fashion, and would thus not be shifted.

In addition to the study cited above, the effects of other substituents on the 3' ends of the invader-" oligonucleotides were investigated in the presence of several different enzymes.

and in the presence of either Mn++ or The effects of these 3' end modifications on the generation of cleaved product are summarized in the following table. All of modifications were made during standard oligonucleotide synthesis by the use of controlled pore glass (CPG) synthesis columns with the listed chemical moiety provided on the support as the synthesis starting residue. All of these CPG materials were obtained from Glen Research Corp. (Sterling, VA).

Fig. 90 provides the structures for the 3' end substituents used in these experiments.

o.

202 Sa* 202 202 TABLE 2 Modification Studies at 3' End of Invader Oligonucleotide 3-End Modification TExtension by IEffect on Invader Ryim ac invadr) 3' phosphate no A:5 inhibits reaction, no detectable activity Glen part N 20-2900-42 3' acridinc yes. A:5 -decrease in activity, Glen part N 20.2973-42 poorly 135 -decrease in activity 10% 13:4 decrease in activity, 101/6 CAl -decrease in activity, C:2 -decrease in activity. 201/.

CA4 decrease in activity, C:3 -decrease in activity. 3' carboxvlate no A: I -decrease in activity -50% activity Glen pant N 20-4090-42 shift in cleavage site C:3 reduces rate. <10% activity 3' nitropyrrole yes A:5 increase in activity, -2X Glen pant 20-2143-42 3' nitroindole yes A:5 decrease in activity. -33% activity Glen part 20-2144-42 3' arabinose yes A:5 decrease in activity, -50% activity Glen pans N 10-4010-90 3'dideoxyUJTP- no A:5 decrease in activity, 40%/ activity fluoresce in linkage no AAl equivalent cleavage activity Glen pan U 20-0002-01 shift in cleavage site C:3 -decrease in activity, -25% activity 3' glycetyl yes, C:3 -decrease in activity, -30% activity Glen part 20-2902-42 very poorly loss of specificity of cleavage (2 tites) 3' amino modifier C7 yes C:3 -decrease in activity. -30% activity Glen parn N 20-2957-42 loss of specificity, multiple sites 3'deoxy. 201-I yes. A:5- decrease in activity, <20% activity Glen pant 20-2104-42 very poorly 11:5 -decrease in activity, activity B:3 -decrease in activity, <20% activity CA equivalent activity C:2- equivalent activity CA4 increase in activity jC:3 -decrease in activity. -40% activity Enzymes A) Cleavase®& DV nuclease B) Cleavase®& BN nuclease C) Pfu FEN- I Condition 1) 4mM MnCi,, 150mM LiCI 2) 4mM MnCI 2 50mM KC1 3) 7.5mM MgCI 2 no monovalent 4) 4mM MgCI 2 50mM KCI 5) 10mM MgOAc, 50mM KCI SeeS

S

S

5 0 50*5 0@ OS S *555 0@ *5 0 0

S

SO..

S

5105 00 5 6

S

5@ 0 0O

SS

203 It can be seen from these data that many different modifications can be used on the 3' end of the InvaderTM oligonucleotide without detriment. In various embodiments of the present invention, such 3' end modifications may be used to block, facilitate, or otherwise alter the hybridization characteristics of the InvaderTM oligonucleotide, to increase discrimination against mismatches, or to increase tolerance of mismatches, or to tighten the association between the Invader T M oligonucleotide and the target nucleic acid). Some substituents may be used to alter the behavior of the enzyme in recognizing and cleaving within the assembled complex.

Altered 3' ends may also be used to prevent extension of the InvaderTM oligonucleotide by either template-dependent or template-independent nucleic acid polymerases. The use of otherwise unmodified dideoxynucleotides without attached dyes or other moieties) are a particularly preferred means of blocking extension of InvaderTM oligonucleotides, because they do not decrease cleavage activity, and they are absolutely unextendable.

EXAMPLE 37 Effect Of Probe Concentration, Temperature And A Stacker Oligonucleotide On The Cleavage Of Miniprobes By InvaderM-Directed Cleavage The stacker oligonucleotides employed to form cleavage structures may serve two purposes in the detection of a nucleic acid target using a miniprobe. The stacker oligonucleotide may help stabilize the interaction of the miniprobe with the target nucleic *..acid. leading to greater accumulation of cleaved probe. In addition, the presence of this oligonucleotide in the complex elongates the duplex downstream of the cleavage site, which may enhance the cleavage activity of some of the enzymes of the present invention. An 25 example of different preferences for the length of this duplex by different structure-specific nucleases is seen in the comparison of the Cleavase® BN nuclease and the Mja FEN-1 nuclease cleavage of 8 bp and 12 bp duplex regions in Fig. 76. Increased affinity of the enzyme for the cleavage structure also results in increased accumulation of cleaved probe o*oo* during reactions done for a set amount of time.

204 The amount of miniprobe binding to the target is also affected by the concentration of the miniprobe in the reaction mixture. Even when a miniprobe is only marginally likely to hybridize when the reaction is performed at temperatures in excess of the expected melting temperature of the probe/target duplex), the amount of probe on the target at any given time can be increased by using high concentrations of the miniprobe.

The need for a stacker oligonucleotide to enhance cleavage of the miniprobe was examined at both low and high probe concentrations. The reactions were carried out in 10 pl of 10 mM HEPES (pH 250 mM KGlu, 4 mM MnC 2 I, containing 100 nM of both the invading (oligonucleotide 135; SEQ ID NO:98) and stacking oligonucleotides (oligonucleotide 147; SEQ ID NO:134) and 100 pM ssM13 DNA. The reactions were overlaid with mineral oil, heated to 90°C for 15 sec then brought to the reaction temperature. Reactions were performed at 350, 400, 45°, 500, 550, 600, and 65°C. The cleavage reactions were initiated by the addition of 1 pl of 100 ng/Il Pfu FEN-I and 1 pl of varying concentrations of Cy-3 labeled 142 miniprobe oligonucleotide (SEQ ID NO:97). Reactions were allowed to proceed for 1 hour and stopped by the addition of 10 pA formaldehyde. One fourth of the total volume of each reaction was loaded onto 20% non-denaturing polyacrylamide gels which were electrophoresed in the reverse direction. Gels were visualized using an Hitachi FMBIO- 100 fluorescent scanner using a 585 nm filter. The fluorescence in each product band was measured and the graph shown in Fig. 91 was created using a Microsoft Excel spreadsheet.

20 The data summarized in Fig. 91 showed that the concentration of the miniprobe had a significant effect on the final measure of product, showing dramatic increases as the concentration was raised. Increases in the concentration of the miniprobe also shifted the 'optimum reaction temperature upward. It is known in the art that the concentration of the complementary strands in a hybridization will affect the apparent T, of the duplex formed 25 between them. More significantly to the methods and compositions of the present invention is the fact that the presence of the stacker oligonucleotide has a profound influence on the cleavage rate of the miniprobe at all probe concentrations. At each of the probe concentrations the presence of the stacker as much as doubled the signal from the cleavage product. This demonstrated the utility of using the stacker oligonucleotide in combination with the miniprobes described herein.

ooo *o 205 EXAMPLE 38 The Presence of A Mismatch In The InvaderTM Oligonucleotide Decreases The Cleavage Activity Of The Cleavase® A/G Nuclease In any nucleic acid detection assay it is of additional benefit if the assay can be made to sensitively detect minor differences between related nucleic acids. In the following experiment, model cleavage substrates were used that were identical except for the presence or absence of a mismatch near the 3' end of the InvaderTM oligonucleotide when hybridized to the model target nucleic acid. The effect of a mismatch in this region on the accumulation of cleaved probe was then assessed.

To demonstrate the effect of the presence of a mismatch in the InvaderT

M

oligonucleotide on the ability of the Cleavase® A/G nuclease to cleave the probe oligonucleotide in an InvaderTM assay the following experiment was conducted. Cleavage of the test oligonucleotide IT-2 (SEQ ID NO:123) in the presence of InvaderTM oligonucleotides IT-1 (SEQ ID NO:124) and IT-IA4 (SEQ ID NO:125). Oligonucleotide IT-1 is fully complementary to the 3' arm of IT-2, whereas oligonucleotide IT-IA4 has a T->A substitution at position 4 from the 3' end that results in an A/A mismatch in the InvaderTM-target duplex. Both the matched and mismatched InvaderTM oligonucleotides would be expected to hybridize at the temperature at which the following reaction was performed. Fig. 92 provides a schematic showing IT-1 annealed to the folded IT-2 structure and showing IT-1A4 annealed to the folded IT-2 structure.

The reactions were conducted as follows. Test oligonucleotide IT-2 (0.1 PM), labeled at the 5' end with fluorescein (Integrated DNA Technologies), was incubated with 0.26 ng/Pl Cleavase® AG in 10 .l of CFLP® buffer with 4 mM MgCI 2 in the presence of 1 pM IT-I or IT-IA4 at 40 0 C for 10 min; a no enzyme control was also run. Samples were overlaid 25 with 15 l Chill-Out® liquid wax to prevent evaporation. Reactions were stopped by addition of 4 ul stop buffer (95% formamide, 20 mM EDTA, 0.02% methyl violet). The cleavage products were separated on a 20% denaturing polyacrylamide gel and analyzed with the FMBIO-100 Image Analyzer (Hitachi) equipped with 505 nm filter. The resulting image is shown in Fig. 93.

In Fig. 93, lane 1 contains reaction products from the no enzyme control and shows the migration of the uncut IT-2 oligonucleotide; lanes 2-4 contain products from reactions containing no InvaderTM oligonucleotide, the IT-1 Invader T M oligonucleotide and the IT-IA4 InvaderTM oligonucleotide, respectively.

206 These data show that cleavage is markedly reduced by the presence of the mismatch, even under conditions in which the mismatch would not be expected to disrupt hybridization.

This demonstrates that the invader oligonucleotide binding region is one of the regions within the complex in which can be used for mismatch detection, as revealed by a drop in the cleavage rate.

EXAMPLE 39 Comparison Of The Activity Of The Pfu FEN-1 And Mja FEN-1 Nucleases In The Invader'" Reaction To compare the activity of the Pfu FEN-I and the Mja FEN-1 nucleases in InvaderTM reaction the following experiment was performed. A test oligonucleotide IT3 (SEQ ID NO:145) that forms an Invader-Target hairpin structure and probe oligonucleotide PRI (SEQ ID NO:127) labeled at the 5' end with fluorescein (Integrated DNA Technologies) were employed in InvaderTM assays using either the Pfu FEN-1 or the Mja FEN-1 nucleases.

The assays were conducted as follows. Pfu FEN-I (13 ng/pl) and Mja FEN-1 ng/pl) (prepared as described in Ex. 28) were incubated with the IT3 (0.1 nM) and PRI (2 and 5 pM) oligonucleotides in 10 pL CFLP® buffer, 4 mM MgCl, 20 mg/ml tRNA at for 41 min. Samples were overlaid with 15 pl Chill-Out® evaporation barrier to prevent evaporation. Reactions were stopped by addition of 70 pl stop buffer (95% formamide, mM EDTA, 0.02% methyl violet). Reaction products (1 pl) were separated on a denaturing polyacrylamide gel, visualized using a fluorescence imager and the bands corresponding to the probe and the product were quantitated. The resulting image is shown in Fig. 94. In Fig. 94, the turnover rate per target per minute is shown below the image for each nuclease at each concentration of probe and target tested.

25 It was demonstrated in Ex. 33 that the use of the Pfu FEN-1 structure-specific nuclease in the InvaderrM-directed cleavage reaction resulted in a faster rate of product accumulation than did the use of the Cleavase® A/G. The data presented here demonstrates that the use of Mja FEN-1 nuclease with the fluorescein labeled probe further increases the amount of product generated by an average of about 50%, demonstrating that, in addition to the Pfu FEN-1 nuclease, the Mja FEN-I nuclease is a preferred structure-specific nuclease for o the detection of nucleic acid targets by the method of the present invention.

207 EXAMPLE Detection Of RNA Target Nucleic Acids Using Miniprobe And Stacker Oligonucleotides Ii adiiunI t to he detection of the Mi3 DNA target material described above, a miniprobe/stacker system was designed to detect the HCV-derived RNA sequences described in Ex. 20. A probe of intermediate length, either a long mid-range or a short standard probe, was also tested. The miniprobe used (oligonucleotide 42-168-1) has the sequence: CCGGTCGTCCTGG-3' (SEQ ID NO:95), the stacker oligonucleotide used (oligonucleotide 32-085) with this miniprobe has the sequence: CAATTCCGGTG TACTACCGGTTCC- 3' (SEQ ID NO:96). The slightly longer probe, used without a stacker (oligonucleotide 42- 088), has the sequence: 5'-TET-CCGGTCGTCCTGGCAA-3' (SEQ ID NO:94). The InvaderTM oligonucleotide used with both probes has the sequence: GTTTATCCAAGAAAGGACCCGGTC-3' (SEQ ID NO:58). The reactions included fmole of target RNA, 10 pmole of the InvaderTM oligonucleotide and 5 pmole of the miniprobe oligonucleotide in 10 tl of buffer containing 10 mM MES, pH 6.5 with 150 mM LiCI, 4 mM MnCI,, 0.05% each Tween-20 and NP-40, and 39 units of RNAsin (Promega Corp., Madison, WI). When used, 10 pmoles of the stacker oligonucleotide was added.

These components were combined, overlaid with ChillOut® evaporation barrier (MJ Research), and warmed to 50 0 C; the reactions were started by the addition of 5 polymerase 20 units of DNAPTth, to a final reaction volume of 10 After 30 minutes at 50 0 C, reactions were stopped by the addition of 8 pl of 95% formamide, 10 mM EDTA and 0.02% methyl violet. The samples were heated to 90 0 C for 1 minute and 2.5 pl of each of these reactions were resolved by electrophoresis through a 20% denaturing polyacrylamide (19:1 cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and the labeled 25 reaction products were visualized using the FMBIO-100 Image Analyzer (Hitachi). The resulting image is shown in Fig. In Fig. 95, lanes 1 and 2 show the products of reactions containing the HCV InvaderTM oligonucleotide and the longer probe (oligonucleotide 42-088), without and with the target RNA present, respectively. Lanes 3, 4, and 5 show the products of reactions containing the InvaderTM oligonucleotide and the shorter probe (oligonucleotide 42-168-1). Lane 3 is a control reaction without target RNA present, while lanes 4 and 5 have the target, but are without or with the stacker oligonucleotide, respectively.

208 i Under these conditions the slightly longer (16 nt) probe oligonucleotide was cleaved quite easily without the help of a stacker oligonucleotide. In contrast, the shorter probe (13 nt) required the presence of the stacker oligonucleotide to produce detectable levels of cleavage. These data show that the miniprobe system of target detection by InvaderTMdirected cleavage is equally applicable to the detection of RNA and DNA targets. In addition, the comparison of the cleavage performance of longer and shorter probes in the absence of a stacker oligonucleotide give one example of the distinction between the performance of the miniprobe/stacker system and the performance of the mid-range and long probes in the detection of nucleic acid targets.

EXAMPLE 41 Construction of Chimerical Structure Specific Nucleases Fig. 70 provides an alignment of the amino acid sequences of several structure-specific nucleases including several each of the FEN-1, XPG and RAD type nucleases. The numbers to the left of each line of sequence refers to the amino acid residue number; portions of the amino acid sequence of some of these proteins were not shown in order to maximize the alignment between proteins. Dashes represent gaps introduced to maximize alignment. From this alignment, it can be seen that the proteins can be roughly divided into blocks of conservation, which may also represent functional regions of the proteins. While not intended as a limitation on the chimeric nucleases of the present invention, these blocks of conservation may be used to select junction sites for the creation of such chimeric proteins.

The Methanococcus jannaschii FEN-1 protein (MJAFEN1.PRO), the Pyrococcus ***furiosus FEN-I protein (PFUFENI.PRO) are shown in the alignment in Fig. 70. These two natural genes were used to demonstrate the creation of chimeric nucleases having different 25 activities than either of the parent nucleases. As known to those of skill in the art, appropriately sited restriction cleavage and ligation would also be a suitable means of creating the nucleases of the present invention. The activities of the parent nucleases on two types of cleavage structures, namely folded structures (See Fig. 71), and invasive structures (See Fig. 30) are demonstrated in the data shown in Figures 96A and 96B, respectively.

These test molecules were digested as described in Ex. 29e. Lanes marked with show cleavage by Pfu FEN-1, while lanes marked with indicate cleavage by Maj FEN-I.

209 In this example, PCR was used to construct complete coding sequences for the chimeric proteins. This is a small subset of the possible combinations. It would also be within common practice in the art to design primers to allow the combination of any fragment of a gene for a nuclease with one or more other nuclease gene fragments, to create further examples of the chimeric nucleases of the present invention. The present invention provides methods, including an activity test, so that the activity of any such chimeric nuclease not explicitly described herein may be determined and characterized. Thus, it is intended that the present invention encompass any chimeric nuclease meeting the requirements of chimeric nucleases, as determined by methods such as the test methods described herein.

To make chimeric nucleases from the M. jannaschii and P. furiosus 5' nuclease genes, homologous parts were PCR amplified using sets of external and internal primers as shown in Fig. 97. In the next step, 5' portions from one gene and a 3' portions from the other gene were joined in pairs by recombinant PCR, such that each combination created a different full size chimerical gene. The resulting coding regions were cloned into the pTrc99A vector and expressed to produce chimerical nucleases. The specific details of construction of each of the chimeric genes shown in Fig. 97 are described below.

a) Construction of chimerical 5' nuclease with M. jannaschii N-terminal portion and P. furiosus C-terminal portion with a junction point at codon 84 (Figure 97g).

A fragment of the pTrc99A vector carrying the M. jannaschii 5' nuclease gene was PCR amplified with TrcFwd (SEQ ID NO:135) and 025-141-02 (SEQ ID NO:136) primers pmole each) in a 50 ;1 reaction using the AdvantageTM cDNA PCR kit (Clonetech), for cycles (92 0 C, 30 s; 55 0 C, 1 min; 72 0 C 1 min) to make an N-terminus-encoding gene fragment (SEQ ID NO:137). The TrcRev (SEQ ID NO:138) and 025-141-01 (SEQ ID NO:139) 25 primers were used to amplify a fragment of the pTrc99A vector carrying the P. furiosus gene to produce a C-terminus encoding gene fragment (SEQ ID NO:140). The PCR products were cleaned with the High Pure PCR Product Purification kit (Boehringer Mannheim, Germany) as described in the manufacturer's protocol and eluted in 100 pl water.

The 025-141-02 (SEQ ID NO:136) primer and the 025-141-01 (SEQ ID NO:139) primer are complementary to each other, so that the PCR fragments created above had the corresponding regions of complementarity on one end. When these fragments are combined in an amplification reaction, the region of complementarity allows the parts to hybridize to each other. to be filled in with the DNA polymerase, and then to be amplified using the outer -210primer pair, TrcFwd (SEQ ID NO:135) and TrcRev (SEQ ID NO:138) in this case, to form one fragment (SEQ ID NO:141). Five pmole of each outer primer was then placed in 50 Vl PCR reaction using the AdvantageTM cDNA PCR kit (Clonetech) as described above. The full length PCR product (SEQ ID NO:141) including the chimerical coding region (positions 1067 of SEQ ID:141) was separated in 1% agarose gel by standard procedures and isolated using the Geneclean II Kit (Bio 101, Vista, CA). The isolated fragment was then cut with NcoI and Pstl restriction enzymes and cloned in pTrc99A vector.

b) Construction of chimerical 5' nuclease with P. furiosus N-terminal portion and M.jannasclii C-terminal portion with a junction point at codon 84 (Fig. 97f).

A fragment of the pTrc99A vector carrying the P. furiosus 5' nuclease gene was PCR amplified with TrcFwd (SEQ ID NO:135) and 025-141-02 (SEQ ID NO:136) primers pmole each) as described above to make an N-terminus-encoding gene fragment (SEQ ID NO:142). The TrcRev (SEQ ID NO:138) and 025-141-01 (SEQ ID NO:139) primers were used to amplify a fragment of the pTrc99A vector carrying the M jannaschii gene to produce a C-terminus encoding gene fragment (SEQ ID NO:143). The fragments were purified and combined in a PCR, as described above to form one fragment (SEQ ID NO: 144), containing the entire chimerical gene (positions 45-1025 of SEQ ID NO:144). This chimerical gene was cut with NcoI and Pstl, and cloned into pTrc99A vector as described in a) above.

c) Construction of chimerical 5' nuclease with P. furiosus N-terminal portiono and M.jannaschii C-terminal portion with a junction point at codon 114 (Fig. 97e).

A fragment of the pTrcPfuHis plasmid was PCR amplified with TrcFwd (SEQ ID NO:135) and 025-164-04 (SEQ ID NO:146) primers (5 pmole each), as described above to 25 make an N-terminus-encoding gene fragment (SEQ ID NO:145). The pTrcPfuHis plasmid was constructed by modifying pTrc99-PFFFEN1 (described in Ex. 28), by adding a histidine tail to facilitate purification. To add this histidine tail, standard primer directed mutagenesis methods were used to insert the coding sequence for six histidine residues between the last amino acid codon of the pTrc99-PFFFEN1 coding region and the stop codon. The resulting plasmid was termed pTrcPfuHis.

The 159-006-01 (SEQ ID NO:148) and 025-164-07 (SEQ ID NO:149) primers were used as described in section a) above, to amplify a fragment of the pTrcMjaHis plasmid to produce a C-terminus encoding gene fragment (SEQ ID NO:147). The pTrcMjaHis plasmid -211 was constructed by modifying pTrc99-MJFENI (described in Ex. 28), by adding a histidine tail to facilitate purification. To add this histidine tail, standard PCR mutagenesis methods were used to insert the coding sequence for six histidine residues between the last amino acid codo uof the pTrc99-MJ}EN 1 coding region and the stop codon. The resulting plasmid was termed pTrcMjaHis. The fragments were purified, and combined by PCR amplification with TrcFwd (SEQ ID NO:135) and 159-006-01 (SEQ ID NO:148) primers in one fragment (SEQ ID NO:150) containing the chimerical gene (positions 45-1043). This chimerical gene was cut with NcoI and Psil, and cloned into pTrc99A vector as described in above.

d) Construction of chimerical 5' nuclease with M. jannaschii N-terminal portion and P. furiosus C-terminal portion with a junction point at codon 148 (Fig. 97d).

A fragment of the pTrc99A vector carrying the M. annaschii 5' nuclease gene was PCR amplified with TrcFwd (SEQ ID NO:135) and 025-119-05 (SEQ ID NO:152) primers, as described above, to make an N-terminus-encoding gene fragment (SEQ ID NO:151). The TrcRev (SEQ ID NO:138) and 025-119-04 (SEQ ID NO:154) primers were used to amplify a fragment of the pTrc99A vector carrying the P. furiosus gene to produce a C-terminus encoding gene fragment (SEQ ID NO:153). The fragments were purified as described above and combined by PCR amplification with the TrcFwd (SEQ ID NO:135) and TrcRev (SEQ ID NO:138) primers into one fragment (SEQ ID NO:155) containing the chimerical gene (positions 45-1067). This chimerical gene was cut with NcoI and Pstl, and cloned into pTrc99A vector as described in above.

e) Construction of chimerical 5' nuclease with P. furiosus N-terminal portion and M.jannaschii C-terminal portion art with a junction point at codon 148 (Fig. 97c).

25 A fragment of the pTrcPfuHis plasmid was PCR amplified with TrcFwd (SEQ ID NO:135) and 025-119-05 (SEQ ID NO:152) primers as described above to make an Nterminus-encoding gene fragment (SEQ ID NO:156). The TrcRev (SEQ ID NO:138) and 025-119-04 (SEQ ID NO:154) primers were used to amplify a fragment of the pTrcMjaHis plasmid to produce a C-terminus encoding gene fragment (SEQ ID NO:157). The fragments were purified as described above and combined by PCR amplification with TrcFwd (SEQ ID NO:135) and TrcRev (SEQ ID NO:138) primers in one fragment (SEQ ID NO:158) containing the chimerical gene (positions 45-1025). This chimerical gene was cut with Ncol and Pstl. and cloned into pTrc99A vector as described in above.

-212 f) Expression and Purification of Chimeras.

All of the chimerical enzymes described above except P. furiosus M. jannaschii construct containing a junction point at the codon 114 Example 41c) were purified as described for Taq DN. The P. furiosus M jannaschii codon 114 chimera with His-tag was purified as described for the 5' nuclease domain BN of Taq Pol I.

g) Activity Characterization of Natural and Chimerical Structure-Specific Nuclease.

All of the chimerical enzymes produced as described above were characterized. In one assay, the enzymes were tested using a mixture of long and short hairpin substrates in the assay system described in Example 29e.

In these tests, reactions were done using 50 ng of each enzyme for 2 min., at 50 0

C.

The results of the analysis are shown in Fig. 98A. In this Figure, the lanes marked and in Figure 98A, indicate reactions with the Pfu and Maj parent enzymes, respectively. The remaining uncut hairpin molecules are visible as two bands at the top of each lane. Each chimeric enzyme tested is represented by reference in Figure 97. For example, the lane marked "97f" shows the cleavage of these test molecules by the chimerical 5' nuclease with the P. furiosus N-terminus and the M jannaschii C-terminus joined at codon 84. The various products of cleavage are seen in the lower portion of each lane. These data show that the chimerical nucleases may display cleavage activities substrate specificities) like either parent 97c and parent Pfu FEN-1 show little cleavage in this test) or distinct from either parent different product profiles).

Similarly, the chimerical enzymes were examined for invasive cleavage activity using the S-60 structure and the P15 oligonucleotide depicted in Fig. 30, as described in Ex. 11.

The results are shown in Fig. 98B. The uncleaved labeled P15 oligonucleotide appears in the i. 25 upper portion of each lane, while the labeled product of cleavage appears in the lower portion.

These results indicate that chimerical enzymes are different in activity and specificity from the original wild-type) M. jannaschii and P. furiosus 5' nucleases.

-213 EXAMPLE 42 Comparison of Digestion of Folded Cleavage Structures With Chimeric Nucleases CFLPIM analysis was applied to a PCR amplified segment derived from E coli 16S rRNA genes. Although bacterial 16S rRNA genes vary throughout the phylogenetic tree, these genes contain segments that are conserved at the species, genus or kingdom level.

These features have been exploited to generate primers containing consensus sequences which flank regions of variability. In prokaryotes, the ribosomal RNA genes are present in 2 to copies, with an average of 7 copies in Escherichia strains. Any PCR amplification produces a mixed population of these genes and is in essence a "multiplex" PCR from that strain.

CFLPT

M analysis represents a composite pattern from the slightly varied rRNA genes within that organism. such that no one particular rRNA sequence is directly responsible for the entire "bar code." As a representative example of an amplicon as described below from the E. coli 16s E. coli rrsE gene is provided (SEQ ID NO:165). Despite the variable nature of these genes, amplification by PCR can be performed between conserved regions of the rRNA genes, so prior knowledge of the entire collection of rRNA sequences for any microbe of interest is not required (See Brow et al., J. Clin. Microbiol., 34:3129 [1996]).

In this Example, the 1638 (5'-AGAGTTTGATCCTGGCTCAG-3')(SEQ ID NO:174) /TET-1659 (5'-CTGCTGCCTCCCGTAGGAGT-3')(SEQ ID NO:175) primer pair was used to amplify an approximately 350 bp fragment of rrsE from genomic DNA derived from E. coli S" 0157: H7 (ATCC #43895). The PCR reactions contained 10 mM Tris-HCI (pH 8.3 at 25 0

C),

mM KC1, 1.5 mM MgCI 2 0.001% w/v gelatin, 60 pM each of dGTP, dATP, dTTP, and dCTP, I i.M of each primer, 25 ng of genomic DNA, and 2.5 units AmpliTaq DNA polymerase. LD in a volume of 100 l. Control reactions that contained no input bacterial 25 genomic DNA were also run to examine the amount of 16S rRNA product produced due to contaminants in the DNA polymerase preparations. The reactions were subjected to 30 cycles of 95°C for 30 sec; 60 0 C for 1 min; 72 0 C for 30 sec; after the last cycle the tubes were cooled to 4°C.

After thermal cycling, the PCR mixtures were treated with E. coli exonuclease I (Exo 1, Amersham) to remove single-stranded partial amplicons and primers. One unit of Exol was added directly to each PCR mixture, and the samples were incubated at 37°C for 20 minutes.

Then, the nuclease was inactivated by heating to 70 0 C for 15 min. The reaction mixtures -214were brought to 2 M NH 4 OAc, and the DNAs were precipitated by the addition of 1 volume of 100% ethanol.

Cleavage reactions comprising 1 pl of TET-labeled PCR products (approximately 100 fmoies) in a total volume of 10 pl containing IX CFLP rM buffer (10 mM MOPS, pH 0.5% each Tween 20 and NP-40) and 0.2 mM MnCI were then conducted. All components except the enzyme were assembled in a volume of 9 pl. The reactions were heated to for 15 sec., cooled to 55 0 C, and the cleavage reactions were started by the addition of 50 ng of enzyme. After 2 minutes at 55 0 C, the reactions were stopped by the addition of 6 pi of a solution containing 95% formamide, 10 mM EDTA and 0.02% methyl violet.

Reaction mixtures were heated at 85°C for 2 min, and were then resolved by electrophoresis through a 10% denaturing polyacrylamide gel (19:1 cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and were visualized using the FMBIO-100 Image Analyzer (Hitachi). The resulting scanned image is shown in Fig. 99. In this Figure, the enzymes used in each digest are indicated at the top of each lane. Cleavase® BN is described in Ex. 2. Lane 2 shows the results of digestion with the Mja FEN-1 parent nuclease, while digests with the chimerical nucleases are indicated by reference to the diagrams in Fig. 97. These data show that the use of each of these nucleases under identical reaction conditions conditions in which the DNA assumes similar folded structures) can produce distinct pattern differences, indicating differences in the specificities of the enzymes.

Thus, each enzyme can provide additional information about the folded structure assumed by a nucleic acid of interest, thereby allowing more accurate comparisons of molecules for identification, genotyping, and/or mutation detection.

These data show that the activities of these enzymes may vary substantially in similar reaction situations. The performance of an optimization panel for an unknown enzyme can 25 help in selection of the optimal enzyme and conditions for a given application. For example, **in the invasive cleavage reactions it is often desirable to choose a combination of nuclease and conditions that perform invasive cleavage, but that do not exhibit activity in the absence of the invader oligonucleotide do not cut a hairpin type substrate).. The optimization panel allows selection of conditions that do not favor hairpin cleavage, such as the use of the Pfu FEN-1 enzyme in a MgCl 2 -containing solution. Conversely, hairpin cleavage is desirable for CFLP-type cleavage, so it is contemplated that reaction conditions be screened accordingly for strength in this activity.

-215 EXAMPLE 43 Characterization of Performance of Structure-Specific Nucleases Two substrates were used to determine the optimal conditions for seven enzymes, Afu, Pfu, Mih and ivija FEN-is, Cleavase® BN, Taq DN and Tth DN. As shown in Figure 107 Panel A, Substrate 25-65-1 (5'-Fluorescein TTTTCGCTGTCTCGCTGAAAGCGAGACAGCGTTT-3'; SEQ ID NO:176) is a stem-loop structure with a 5' arm labeled at its 5' end with fluorescein. As shown in Figure 107 Panel B, substrate 25-184-5 (Invader-like "IT" test substrate") CGAGCGTCTTTG-3'; SEQ ID NO:177) is a substrate with an upstream primer adjacent to the 5' fluoroscein labled arm; this mimics an invader oligo and target Standard reactions contained 2 pM labeled substrate, 10 mM MOPS, pH7.5, 0.05% TWEEN 20, 0.05% 20 pg/ml tRNA (Sigma R-5636) and 2 mM MgCl2 or 2 mM MnCI,. Ten il reactions were heated to 90 0 C for 15 seconds in the absence of enzyme and divalent cation.

after which the reactions were cooled to room temperature and enzyme was added. Reactions were heated to 50 0 C for 20 seconds and divalent cation was then added to start the reaction.

The incubation time varied from 1 minute to 1 hour depending on the particular enzyme/substrate combination. Reaction times were adjusted so that less than 25% of the substrate was cleaved during the incubation. Reactions were stopped with the addition of pl of 95% formamide. 20 mM EDTA, methyl violet. One pl of each reaction was electrophoresed on a 20% denaturing acrylamide gel and then scanned on an FMBIO 100 scanner (Hitachi).

Divalent cation titrations varied MgCI, or MnCI, from 0.25 mM to 7 mM under otherwise standard conditions. Salt titrations varied KCI from 0 mM to 200 mM or 400 mM 25 for salt tolerant enzymes under otherwise standard conditions. For temperature titrations, reactions with Cleavase® BN and the FEN-1 enzymes contained 50 mM KCI and 4 mM MgCI 2 or MnCl,. Temperature titrations with Taq DN and Tth DN contained 200 mM KCI and 4 mM MgCI 2 or MnCI,. Temperature was varied from 40 0 C to 85 0 C in 5 or 10 degree increments depending on the particular enzyme used.

The results are shown in Figures 100-106. Figure 100 shows the results for Cleavase® BN, while Figure 101 shows the results for Taq DN, Figure 102 shows the results for Tth DN, Figure 103 shows the results for Pfu FEN-, Figure 104 shows the results for Mja FEN-, Figure 105 shows the results for Afu FEN-1, and Figure 106 shows the results for Mth -216-

I

FEN-1. In each of the Panels within these Figures, the activity of the enzyme is defined as cleavages per molecule of enzyme per minute. Panels marked "IT" refer to cleavage of the 25-184-5 structure (SEQ ID NO:177; Fig. 107B), which mimics an invader oligo/target DNA structure, while Panels marked with "hairpin" refer to cleavage of the 25-65-1 structure (SEQ ID NO:176; Fig. 107A), which indicates activity on folded cleavage structures.

In each of these Figures, Panel A shows the results from reactions containing 2 mM MgCI, and the IT substrate as described in the text, with KCI varied as indicated; Panel B shows the results from reactions containing 2 mM MnC1 2 and the IT substrate as described in the text, with KCI varied as indicated; Panel C shows the results from reactions containing 2 mM MgCI, and the hairpin substrate as described in the text, with KCI varied as indicated; Panel D shows the results from reactions containing 2 mM MnCI, and the hairpin substrate as described in the text, with KCI varied as indicated; Panel E shows the results from reactions containing the IT substrate as described in the text, with MgC 2 varied as indicated; Panel F shows the results from reactions containing the IT substrate as described in the text, with MnCI, varied as indicated; Panel G shows the results from reactions containing the hairpin substrate as described in the text, with MgCIl varied as indicated; Panel H shows the results from reactions containing the hairpin substrate as described in the text, with MnC1, varied as indicated; Panel I shows the results from reactions containing the IT substrate, 4 mM MgCI, mM KCI (Afu FEN-1, Pfu FEN-1, Mja FEN-1, Mth FEN-l, and Cleavase® FN) or 200 mM KCI (Taq DN and Tth DN) as described in the text, with the temperature varied as indicated; and Panel J shows the results from reactions containing the IT substrate, 4 mM MnCI,, and 50 mM KCI (Afu FEN-1, Pfu FEN-1, Mja FEN-1, Mth FEN-1, and Cleavase® BN) or 200 mM KC1 (Taq DN and Tth DN) as described in the text, with the temperature varied as indicated. It is noted that some of these Figures 101, 102, 103, and 105) do not show each of the above-named panels A-J.

From the above it is clear that the invention provides reagents and methods to permit the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The Invaderm-directed cleavage reaction of the present invention provides an ideal direct detection method that combines the advantages of the direct detection assays easy quantification and minimal risk of carry-over contamination) with the specificity provided by a dual or tri oligonucleotide hybridization assay.

o o 217 All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scupe a iud spirii of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

*oo •••go 218 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Kaiser, Michael W.

Lyamichev, Victor I.

Lyamichev, Natasha (ii) TITLE OF INVENTION: Improved Cleavage Agents (iii) NUMBER OF SEQUENCES: 188 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Medlen Carroll, LLP STREET: 220 Montgomery Street, Suite 2200 CITY: San Francisco STATE: California COUNTRY: United States Of America ZIP: 94104 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE: 02-DEC-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/ FILING DATE: 29-NOV-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/682,853 FILING DATE: 12-JUL-1996 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/599,491 FILING DATE: 24-JAN-1996 (viii) ATTORNEY/AGENT INFORMATION: S NAME: McKnight, Kamrin T.

REGISTRATION NUMBER: 38,230 REFERENCE/DOCKET NUMBER: FORS-03104 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (415) 705-8410 TELEFAX: (415) 397-8338 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2506 base pairs TYPE: nucleic acid STRANDEDNESS: double o TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG 120 GTGCAGGCGG TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC 180 -219-

GCGGTGATCG

TACAAGGCGG

GAGCTGGTGG

rCr,

GCCGACAAAG

TACCTCATCA

GACTACCGGG

GAGAAGACGG

CTGGACCGGC

CTCTCCTGGG

AGGCGGGAGC

CTCCTCCACG

CCGCCGGAAG

CTTCTGGCCC

GCCCTCAGGG

CTGAGGGAAG

GACCCTTCCA

GAGGCGGGGG

GAGGGGGAGG

CTGGCCCACA

CTGGAGGTGG

CCCTTCAACC

CCCGCCATCG

GCCCTCCGCG

CTGAAGAGCA

CACACCCGCT

CTCCAGAACA

GAGGAGGGGT

CACCTCTCCG

GAGACCGCCA

GCGGCCAAGA

GAGCTAGCCA

CCCAAGGTGC

GAGACCCTCT

TGGTCTTTGA

GCCGGGCCCC

ACCTCCTGGG

G.C07GGCCAA

ACCTTTACCA

CCCCGGCCTG

CCCTGACCGG

CGAGGAAGCT

TGAAGCCCGC

ACCTGGCCAA

CCGACCGGGA

AGTTCGGCCT

GGGCCTTCGT

TGGCCGCCGC

ACCTGAAGGA

GCCTTGGCCT

ACACCACCCC

AGCGGGCCGC

AGAGGCTCCT

TGGAGGCCAC

CCGAGGAGAT

TCAACTCCCG

GCAAGACGGA

AGGCCCACCC

CCTACATTGA

TCAACCAGAC

TCCCCGTCCG

GGCTATTGGT

GCGACGAGAA

GCTGGATGTT

CCATCAACTT

TCCCTACGA

GGGCCTGGAT

TCGGCCGCCG

CGCCAAGGCC

CACGCCGGAG

GCTGGCGCGC

UAAGGCGGAA

GCTCCTTTCC

GCTTTGGGAA

GGACGAGTCC

TCTGGAGGAG

CATCCGGGAG

GGTGCGCACC

GAGGCTTAGG

TCTGGAAAGC

GGGCTTTGTG

CAGGGGGGGC

GGCGCGGGGG

CCCGCCCGGC

CGAGGGGGTG

CCTTTCCGAG

TTGGCTTTAC

GGGGGTGCGC

CGCCCGCCTC

GGACCAGCTG

GAAGACCGGC

CATCGTGGAG

CCCCTTGCCG

GGCCACGGCC

CACCCCGCTT

GGCCCTGGAC

CCTGATCCGG

CGGCGTCCCC

CGGGGTCCTC

GGAGGCCCAG

TGAGAAGACC

CCGCTACGTG

CCCTCCTTCC

GACTTTCCCC

CTCGAGGTCC

AAGGAGGGCT

GACCGCATCC

AAGTACGGCC

GACAACCTTC

TGGGGGAGCC

AAGATCCTGG

GACCTGCCCC

GCCTTTCTCG

CC CAAGGC CC

CTTTCCCGCA

CGGGTCCACC

CTTCTCGCCA

GACGACCCCA

GCCCGGCGCT

AGGCTCTTCG

CGGGAGGTGG

CTGGACGTGG

GAGGCCGAGG

GAAAGGGTCC

AAGCGCTCCA

AAGATCCTGC

GACCTCATCC

ACGGGCAGGC

GGGCAGAGGA

TATAGCCAGA

GTCTTCCAGG

CGGGAGGCCG

TACGGCATGT

GCCTTCATTG

CTGGAGGAGG

CCAGACCTAG

GCCACGAGGC

GGCAACTCGC

CGGGCTACGA

ACGAGGTCCG

ACGTCCTCCA

TGAGGCCCGA

CCGGGGTCAA

TGGAAGCCCT

CCCACATGGA

TGGAGGTGGA

AGAGGCTTGA

TGGAGGAGGC

AGGAGCCCAT

GGGCCCCCGA

AAGACCTGAG

TGCTCCTCGC

ACGGCGGGGA

CCAACCTGTG

AGAGGCCCCT

CCTATCTCAG

TCTTCCGCCT

TCTTTGACGA

CCAGCGCCGC

AGTACCGGGA

ACCCCAGGAC

TAAGTAGCTC

TCCGCCGGGC

TAGAGCTCAG

AGGGGCGGGA

TGGACCCCCT

CGGCCCACCG

AGCGCTACTT

GCAGGAGGCG

AGGCCCGGGT

AGGGCACCGC

CTACGGGGGG

CCTCA'rCAAC-

GGCGGACGAC

CATCCTCACC

CCCCGAGGGG

CCAGTGGGCC

GGGCATCGGG

CCTCAAGAAC

CGATCTGA-AG

CTTCGCCAAA

GTTTGGCAGC

CCCCTGGCCC

GTGGGCCGAT

GCCTTATAAA

CGTTCTGGCC

CTACCI'CCTG

GTGGACGGAG

GGGGAGGCTT

TTCCGCTGTC

GGCCTTGTCC

GGCCGGCCAC

GCTAGGGCT'r

CGTCCTGGAG

GCTCACCA-AG

GGGCCGCCTC

CGATCCCAAC

CTTCATCGCC

GGTGCTGGCC

CATCCACACG

GATGCGCCGG

CCTCTCCCAG

TCAGAGCTTC

GGGGTACGTG

GAAGAGCGTG

CGCCGACCTC

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 9*

S

A

5 5 .5 S S S SS CGGGAGGCGG CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC 220 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AA.ATGGGGGC CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG TATCCCCTGG CCGTGCCCCT GGAGGTGGAG G*.1'UGGATAG GGGAGGACTG GCTCTCCGCC AAGGAGTGAT ACCACC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 2496 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 2340 2400 2460 2506 0 S S *5S0 S S *5 S

S

555 5

ATGGCGATGC

CTGGCCTACC

CAGGCGGTCT

GTGGTGGTGG

AAGGCGGGCC

TTGGTGGACC

CTGGCCACCC

GACCGCGACC

CTGATCACCC

TACCGGGCCC

AAGACCGCCC

GACCAGGTGA

TCCCGGAAGC

CGCACACCCA

CTCCACGAGT

CCGGAAGGGG

CTGGCCCTGG

CTGAGGGACC

CGGGAGGGCC

CCCTCCAACA

GCGGGGGAGA

GGAGAAGAAC

GCCCGGATGG

GAGGTGGAGG

TTCAACCTCA

TTCCCCTCTT

GCACCTTCT T

ACGGCTTCGC

TCTTGACGC

GGGCCCCCAC

TCCTAGGCCT

TGGCCAAGCG

TCTACCAGCT

CGGCGTGGCT

TGGCGGGGGA

AGAGGCTCAT

AGCCCTCCTT

TTTCCCAGGT

ACCTGGAGGG

TCGGCCTCCT

CTTTTTTGGG

CTGGGGCGTG

TTAAGGGGGT

TGGACCTCTT

CCACCCCTGA

GGGCCCTCCT

GCCTGCTTTG

AGGCCACGGG

CGGAGGTGCG

ACTCCCGCGA

TGAGCCCAAA

TGCCCTCAAG

CAAAAGCCTC

CAAGGCCCCC

CCCGGAGGAC

TGTGCGGCTG

GGCGGAAAAG

CCTTTCGGAG

TTACGAGAAG

CCCCTCGGAT

CCGCGAGTGG

GCGGGAGAAG

GCACACTGAC

TCTGCGGGCT

GGAGGGGCCG

CTI'TTCCTTT

GGAGGGGCGC

GCGGGGAATC

CCCAGAGGAC

GGGGGTGGCC

GGCCGAGCGC

GCT TT.ACGAG

GGTCCGGCTG

CCAGCTGGAG

CCAGC1TGGAG

GGCCGCGTGC

GGCCTCACCA

CTCAAGGCCC

TCCTTCCGCC

TTTCCCCGGC

GAGGTTCCCG

GAGGGGTACG

CGCATCGCCA

TACGGCCTGC

AACATCCCCG

GGGAGCCTGG

CTCCAGGCGG

CTGCCCCTGG

TTTTTGGAGC

AAGGCGGCAG

TCCCGTCCCG

CTCCATCGGG

CTGGCCAAGG

GACCCCATGC

CGGCGTTACG

CTCTTCCAGA

GAGGTGGAGA

GACGTGGCCT

GAGGAGGTCT

CGGGTGCTCT

TCCTGGTGGA

CCAGCCGCGG

TGAAGGAGGA

ACGAGGCCTA

AGCTGGCCCT

GCTTGAGGC

AGGTGCGCAT

TCCTCCACCC

GCCCGGAGCA

GGGTGAAGGG

AAAACCTCTT

GCATGGAGGC

AGGTGGACTT

GGTTGGAGTT

AGGAGGCCCC

AGCCCATGTG

CACAAGACCC

ACCTGGCGGT

TCCTGGCCTA

GGGGGGAGTG

CCCTAAAGGA

AGCCGCTTTC

ACCTCCAGGC

TCCGCCTGGC

TTGACGAGCT

CGGCCACCAC

CGAACCCGTT

CGGGGACGTG

CGAGGCCTAC

CATCA-AGGAG

GGACGACGTG

CCTCACTGCC

TGAGGGGTAC

GTGGGTGGAC

CATCGGGGAG

CCAGCACCTG

CCTGGCCCTT

CGGGAGGCGC

TGGAAGCCTC

CTGGCCCCCT

GGCCGAGCTT

CCTTAGGGGC

TTTGGCCCTG

CCTTCTGGAC

GACGGAGGAT

GCGCCTTAAG

CCGGGTGTTG

CCTCTCCCTG

CGGCCACCCC

GGGCCTGCCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 SS S 5 0* S 0 @0 S 50 221 GCCATCGGCA AGACGGAGAA GACGGGGAAA CGCTCCACCA GCGCTGCCGT GCTGGAGGCC

CTGCGAGAGG

AAGAACACCT

CAGAACATCC

GAGGGCTGGG

CTCTCCGGGG

ACCGCCAGCT

GCCAAGACCA

CTTTCCATCC

AAGGTGCGGG

ACCCTCTTCG

GAGGCGGCGG

AAGCTGGCCA

CAGGTGCACG

TTGGCCA.AGG

GGCCTGGGGG

CCCACCCCAT

ACATAGACCC

CCGTGCGCAC

TGCTGGTGGT

ACGAGAACCT

GGATGTTCGG

TCAACTTCGG

CCTACGAGGA

CCTGGATTGA

GCCGCCGGCG

AGCGCATGGC

TGGTGCGGCT

ACGAGCTGGT

AGGTCATGGA

AGGACTGGCT

CGTGGACCGC

CCTGCCCGCC

C.rCC.xCCACG

CCCTCTGGGC

CTTGGACTAC

GATCCGGGTC

CGTTTCCCCC

GGTGCTCTAC

GGCGGTGGCC

GGGGACCCTC

CTATGTGCCC

CTTCA.ACATG

TTTCCCCCGG

CCTCGAGGCC

GGGGGTCTGG

CTCCGCCAAG

ATCCTGCAGT

CTGGTCCACC

UGCAGGCTTT

CAGCGCATCC

AGCCAGATTG

TTTCAGGAGG

GAAGGGGTAG

GGCATGTCCG

TTCATTGAGC

GAGGAGGGCC

GACCTCAACG

CCGGTCCAGG

CTTCAGGAAC

CCCAAGGACC

CCCCTGCAGG

GAGTAG

ACCGGGAGCT

CCAAGACCGG

CCAGCTCCGA

GCCGAGCCTT

AGCTTCGGGT

GGAGGGACAT

ACCCTCTGAT

CCCACCGCCT

GCTACTTCCA

GCCGGCGGGG

CCCGGGTGAA

GCACCGCCGC

TGGGGGCGAG

GGGCGGAGAG

TGCCCCTGGA

CACCAAGCTC

CCGGCTCCAC

CCCCAACCTG

CGTGGCCGAG

CCTGGCCCAC

CCACACCCAG

GCGCCGGGCG

CTCCGGGGAG

GAGCTACCCC

GTATGTGGAG

GAGCGTGCGC

CGACCTCATG

GATGCTTTTG

GGTAGCCGCT

GGTGGAGGTG

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2496

S.

*000 0 0 0 0 *0S*

OSS@

@0 @0 S 0

S

SO

0

S

000*0*

S

0~Se

S

0~S0 0000*0 0 000000 0 00 50 0 S 0 00 S 0~e

SO

INFORMATION FOR SEQ ID NO:3: SEQUENCE CH{ARACTERISTICS: LENGTH: 2504 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGGAGGCGA TGCTTCCGCT CTTTGAACCC AAAGacCCOn

CACCTGGCCT

GTGCAGGCGG

AAGGCCGTCT

GCCTACAAGG

A.AGGAGCTGG

GACGTTCTCG

ACCGCCGACC

GGCCACCTCA

GTGGACTTCC

GGGGAGAAGA

AACCTGGACC

ACCGCACCTT

TCTACGGCTT

TCGTGGTCTT

CGGGGAGGGC

TGGACCTCCT

CCACCCTGC

GCGACCTCTA

TCACCCCGGA

GCGCCCTCGT

CCGCCCTCA.A

GGGTAAAGCC

CTTCGCCCTG

CGCCAAGAGC

TGACGCCAAG

CCCGACCCCC

GGGGTTTACC

CAAGAAGGCG

CCAACTCGTC

GTGGCTTGG

GGGGGACCCC

GCTCCTCAAG

AGAAAACGTC

A.AGGGCCTCA

CTCCTCAAGG

GCCCCCTCCT

GAGGACTTCC

CGCCTCGACG

GAAAAGGAGG

TCCGACCGCG

GAGAAGTACG

TCCGACAACC

GAGTGGGGAA

CGGGAGAAGA

TCCTCCTGGT

CCACGAGCCG

CCCTGA-AGGA

TCCGCCACGA

CCCGGCAGCT

TCCCCGGCTA

GGTACGAGGT

TCGCCGTCCT

GCCTCAGGCC

TCCCCGGGGT

GCCTGGAAAA

TCAAGGCCCA

GGACGGCCAC

GGGCGAACCG

GGACGGGTAC

GGCCTACGAG

CGCCCTCATC

CGAGGCGGAC

GCGCATCCTC

CCACCCCGAG

GGAGCAGTGG

CAAGGGCATC

CCTCCTCA.AG

CCTGGAAGAC

-222

CTCAGGCTCT

GCCCAGGGGC

GGCAGCCTCC

GCGGAGCTTA

TTGGCGGGGC

TTGGCCTCGA

CTCCTGGACC

ACGGAGGACG

CGCCTCGAGG

CGGGTCCTGG

C=TTCCCTGG

GGCCACCCCT

AGGCTTCCCG

CTGGAGGCCC

ACCAAGCTCA

CGCCTCCACA

CCCAACCTGC

GTGGCCGAGG

CTCGCCCACC

CACACCCAGA

CGCCGGGCGG

TCCCAGGAGC

GCTTCCCCAA

ACGTGGAAAC

GCGTCAGGGA

ACCTCATGAA

TGCTCCTCCA

TGGCGGCTTT

TGGAGGTGGG

CCTGGAGCT

GGGAGCCCGA

TCCACGAGTT

AAGCCCTGGC

TAAAGGACCT

GGGAGGGGCT

CCTCCAACAC

CCGCCCACCG

GGGAGGAGAA

CCCACATGGA

AGCTT-GCGGA

TCAACCTCAA

CCTTGGGGAA

TACGGGAGGC

AGAACACCTA

CCCGCTTCAA

AGAACATCCC

CGGGTTGGGC

TCTCCGGGGA

CCGCAAGCTG

CCAAGACGGT

TTGCCATCCC

GGTGCGGGCC

CCTCTTrCGGA

GGCCGCGGAG

GCTCGCCATG

GGTCCACGAC

GGCCAAGGAG

GATGGGGGAG

CTCCCGGGTG

CCGGGAGGGG

CGGCCTCCTG

CT-*I*LU'IGGGC

CGCCTGCAGG

CAAGGAGGTC

AGACCTCGTG

CACCCCCGAG

GGCCCTCCTC

GCTCCTTTGG

GGCCACCGGG

GGAGATCCGC

CTCCCGGGAC

GACGCAAAAG

CCACCCCATC

CGTGGACCCC

CCAGACGGCC

CGTCCGCACC

GTTGGTGGCC

CGAAAACCTG

GATGTTCGGC

GAACTTCGGC

CTACGAGGAG

TGGATAGAAA

AGAAGGCGCT

CGCATGGCCT

GTGAAGCTCT

GAGCTCCTCC

GCCATGGAGA

GACTGGCTTT

CGCACCGACC

C'ITAGGGCCT

GAGGCCCCCG

TTCGTCCTCT

GACGGCCGGG

CGGGGCCTCC

CCCGGGGACG

GGGGTGGCGC

TCGGAGAGGC

CTCTACCACG

GTACGGCTGG

CGCCTCGAGG

CAGCTGGAAA

ACAGGCAAGC

GTGGAGAAGA

CTCCCAAGCC

ACGGCCACGG

CCCTTGGGCC

CTGGACTATA

ATCAGGGTCT

GTCCCCCCGG

GTCCTCTACG

GCGGTGGCCT

AGACCCTGGA

ACGTGCCCGA

TCAACATGCC

TCCCCCGCCT

TGGAGGCCCC

AGGCCTATCC

CCGCCAAGGG

TCCCCCTGGA

TCCTGGAGAG

CCCCCCTGGA

CCCGCCCCGA

TGCACCGGGC

TCGCCAAGGA

ACCCCATGCT

GGCGCTACGG

TCCATCGGAA

AGGTGGAAAA

ACGTGGCCTA

AGGAGGTCTr

GGGTGCTCTT

GCTCCACCAG

TCCTCCAGCA

TCGTCCACCC

GGAGGCTTAG

AGAGGATCCG

GCCAGATAGA

TCCAGGAGGG

AGGCCGTGGA

GCATGTCCGC

TTATAGAGGC

GGAGGGGAGG

CC'rCAACGCC

CGTCCAGGGC

CCGGGAGATG

CCAAGCGCGG

CCTCGCCGTG

TTAG

GGTGGACCTC

GCTGGAGTTC

GGAGGC CCC C

GCCCATGTGG

AGCAGACCCC

CCTCGCCGTC

CCTCGCCTAC

GGGGGAGTGG

CCTCCTTAAG

GCCCCTCTCC

CCTTCAGGCC

CCGCTTGGCG

TGACGAGCTT

CGCCGCGGTG

CCGGGAGCTC

GAGGACGGGC

TAGCTCCGAC

CCGGGCCTTC

GCTCCGCGTC

GAAGGACATC

CCCCCTGATG

CCATAGGCTC

TACTTCCAAA

AAGCGGGGCT

CGGGTGAAGA

ACCGCCGCCG

GGGGCCCGCA

GCCGAGGAGG

CCCCTGGAGG

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2504 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 832 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 223 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Arg Gly Met Val Leu Lys Val1 Tyr Al a Val1 Ala Leu 145 Tyr Asp Leu Glu Lys 225 Leu Asp Leu Glu Al a 305 Leu Glu Ala Asp Thr Ser Phe Lys Leu Pro Glu 130 Tyr Leu Gin Pro Gl u 210 Pro Ser Phe Glu Ser 290 Phe Leu Pro Lys Gly Thi- Leu Aso Ala Ile Gly 115 Lys Gin Ile Trp Gly 195 Ti-p Ala Ti-p Al a Arg 275 Pro Val1 Al a Tyr Asp 355 His Ser Leu Ala Gly Lys 100 Tyr Giu Leu Thr Al a 180 Val1 Gly Ile Asp Lys 260 Leu Lys Gly Leu Lys 340 Leu Leu His Arg Lys Lys Arg Glu Glu Gly Leu Pro 165 Asp Lys Ser Arg Leu 245 Arg Glu Ala Phe Al a 325 Al a Ser Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Leu Gly Al a Al a 70 Al a Leu Al a Tyr Ser 150 Al a Tvr Gly Leu Glu 230 Al a Arg Phe Leu Val1 310 Ala Leu Val1 Ala Glu Leu 55 Pro Pro Val1 Asp Glu 135 Asp Ti-p Arg Ile Glu 215 Lys Lys Glu Gly Glu 295 Leu Al a Ar g Leu Tyr Pro 40 Lys Ser Thi- Asp Asp 120 Val1 Arg Leu Ala Gly 200 Al a Ile Val Pro Ser 280 Glu Ser Arg Asp Al a Arg Val1 Giu Phe Pro Leu 105 Val1 Arg Ile Trp, Leu 185 Ci u Leu Leu Arg Asp 265 Leu Ala Arg Gly Leu 345 Leu Thr Gln Asp Arg Glu 90 Leu Leu Ile His Glu 170 Th- Lys Leu Al a Thr 250 Arg Leu Pro Lys Gly 330 Lys Arg Phe Al a Gly His 75 Asp Gly Al a Leu Val1 155 Lys Gly Thr Lys His 235 Asp Giu His 'r-p Glu 315 Arg Glu Glu Ala Tyr Al a Ala Pro Ala Leu 125 Al a His Gly Glu Arg 205 Leu Asp Pro Leu Phe 285 Pro Met His Arg Leu 365 Leu Gly Val1 Tyr Arg Arg 110 Ala Asp Pro Leu Ser 190 Lys Asp Asp Leu Arg 270 Gly Pro Ti-p Arg Gly 350 Gly Lys Phe Ile Gly Gin Leu Lys Lys Glu Arg 175 Asp Leu Arg Leu C lu 255 Ala Leu Glu Al a Ala 335 Leu Leu r(~1 Ala Val1 Gly Leu Giu Lys Asp Gi y 160 Pro Asn Leu Leu Lys 240 Val Phe Leu Gly Asp 320 Pro Leu Pro 360 224 Pro Thr 385 Glu Trp Val1 Val1 Glu 465 Pro Gi u Ser Val1 Tyr 545 His Ser Arg Leu Asp 625 Glu Leu Met Ala Ala 705 Glu Asp Glu Glu Leu 420 Pro Asp Ala Leu Leu 500 Al a Ile Pro Phe Asn 580 Arg Ser Leu Ser Arg 660 His Phe Glu Phe Al a Arg Ser Arg 425 Leu Arg Glu Gln Lys 505 Ala Glu Ile Thr Pro 585 Glu Arg Gln Val Ile 665 Glu Phe Glu Tyr Leu 380 Gl y Leu Trp Met Ser 460 Arg Arg Lys Glu Lys 540 Arg Gly Thr Trp Ala 620 Arg Glu Gly Ile Phe 700 Arg Asp 225 Val Lys Ser Val Arg Giu Ala Ala Glu Arg Met Ala Phe Asn- Met Pro 740 745 750 Val Gin Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Le Gl Gl ie L Gy A.-La Arg met Leu Leu Gin Val His 770 775 780 Asp Giu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Giu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Giu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Giu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 831 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Met Leu Pro Leu Phe Giu Pro Lys Gly Arg Val Leu Leu Val 1 5 10 Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly Leu 25 Thr Thr Ser Arg Giy Glu Pro Val Gin Ala Val Tyr Gly Phe Ala Lys 40 Ser Leu Leu Lys Ala Leu Lys Giu Asp Gly Asp Val Val Val Val Val 55 Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr *65 70 75 Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gin Leu Ala ***8590 *Leu Ile Lys Giu Leu Val Asp Leu Leu Gly Leu Val Arg Leu Giu Val 100 105 110 Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Arg Ala 115 120 125 Glu Lys Glu Gly Tyr Glu Val Az-g Ile Leu Thr Ala Asp Arg Asp Leu 130 135 140 Tyr Gin Leu Leu Ser Glu Arg Ile Ala Ile Leu His Pro Giu Gly Tyr 145 150 155 160 Leu Ile Thr Pro Ala Trp Leu Tyr Glu Lys Tyr Gly Leu Arg Pro Glu 165 170 175 Gin Trp Val Asp Tyr Arg Ala Leu Ala Gly Asp Pro Ser Asp Asn Ile 180 185 190 **:Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Gin Arg Leu Ile Arg 195 200 205 226 Giu Trp Gly Ser Leu Glu Asn Leu Phe Gin His Leu Asp Gin Val Lys 210 215 220 Pro Ser Leu Arg Glu Lys Leu Gin Ala Gly Met Giu Ala Leu Ala Leu 225 230 235 240 Ser Arg Lys Leu Ser Gin Val His Thr Asp) Leu Pro Leu Glu Val Asp 245 250 255 Phe Gly Arg Arg Arg Thr Pro Asn Leu Giu Gly Leu Arg Ala Phe Leu 260 265 270 Glu Arg Leu Giu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu 275 280 285 Gly Pro Lys Ala Ala Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala 290 295 300 Phe Leu Gly Phe Ser Phe Ser Arg Pro Glu Pro Met Trp Ala Glu Leu 305 310 315 320 Leu Ala Leu Ala Gly Ala Trp Giu Gly Arg Leu His Arg Ala Gin Asp 325 330 335 Pro Leu Arg Gly Leu Arg Asp Leu Lys Gly Val Arg Gly Ile Leu Ala 340 345 350 Lys Asp Leu Ala Val Leu Ala Leu Arg Glu Gly Leu Asp Leu Phe Pro 355 360 365 Giu Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr 370 375 380 Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Giu Asp 385 390 395 400 Ala Gly Glu Arg Ala Leu Leu Ala Glu Arg Leu Phe Gln Thr Leu Lys 405 410 415 Giu Arg Leu Lys Gly Giu Glu Arg Leu Leu Trp Leu Tyr Glu Glu Val 420 425 430 Giu Lys Pro Leu Ser Arg Val Leu Ala Arg Met Glu Ala Thr Gly Val 435 440 445 Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Giu Val Giu Ala *450 455 460 Glu Val Arg Gin Leu Glu Glu Glu Val Phe Arg Leu Ala Gly His Pro 465 470 475 480 *Phe Asn Leu Asn Ser Arg Asp Gin Leu Glu Arg Val Leu Phe Asp Glu 485 490 495 Leu Gly Leu Pro Ala Ile Gly Lys Thr Giu Lys Thr Gly Lys Arg Ser 500 505 510 Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Aia His Pro Ile Val .515 520 525 Asp Ar Ile Leu Gin Tyr Arg Glu Leu Thr Lys Leu Lys Asn Thr Tyr 530 535 540 Ile Asp Pro Leu Pro Ala Leu Val His Pro Lys Thr Gly Arg Leu His .545 550 555 560 *Thr Arg Phe Asn Gin Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser 565 570 575 227 Asp Pro Asn Leu 580 Gin Asn Ile Pro Val Arg Thr Pro Leu 585 Gly Gin Arg 590 Giu 600 Arg Gin Val1 Ile Glu 680 Phe Giu Tyr Giu Met 760 Aia Lys Giy Glu Giu Val Giu Vai Giy Leu Giy 820 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 834 amino acids TYPE: aminoaicid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Aia Met Leu Pro Leu Phe Giu Pro Lys Giy Arg Val Leu Leu 1 5 10 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly 20 25 Leu Thr Thr Ser Arg Giy Glu Pro Val Gin Ala Val Tyr Giy Phe Ala 40 228 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Lys Ala Val Phe Leu Leu 410 229 Ser Glu Arg Leu His Arg 415 Asn Leu Leu Lys Arg Leu Glu Gly Glu Giu Lys Leu Leu 420 His Thr Leu 465 Gly Phe Lys Pro Asn 545 Arg Ser Gly Val Ser 625 His Asp Tyr Glu Val 705 Tyr Ala Met Lys Glu Gly 450 Ala His Asp Arg Ile 530 Thr Leu Ser Gin Ala 610 Gly Thr Pro Gly Glu 690 Arg Val Arg Pro Leu 770 Val 435 Val Glu Pro Glu Ser 515 Val Tyr His Ser Arg 595 Leu Asp Gin Leu Met 675 Ala Ala Glu Jal Ja1 755 Phe Glu Arn Glu Phe Leu 500 Thr Glu Val Thr Asp 580 Ile Asp Glu Thr Met 660 Ser Val Trp Thr Lys 740 Gin Pro Lys Ile Asn 485 Arg Ser Lys Asp Arg 565 Pro Arg Tyr Asn Ala 645 Arg Ala Ala Ile Leu 725 Ser 3iy Arg Pro Arg 470 Leu Leu Ala Ile Pro 550 Phe Asn Arg Ser Leu 630 Ser Arg His Phe Glu 710 Phe Va1 Thr Leu Leu 455 Arg Asn Pro Ala Leu 535 Leu Asn Leu Ala Gin 615 Ile Trp Ala Arg Ile 695 Lys Gly Arg Ala Arg 775 Ser 440 Ala Leu Ser Ala Val 520 Gin Pro Gin Gin Phe 600 Ile Arg Met Ala Leu 680 Glu Thr Arg 31u Ala 760 31u 425 Arg Tyr Glu Arg Leu 505 Leu His Ser Thr Asn 585 Val Glu Val Phe Lys 665 Ser Arg Leu Arg Ala 745 Asp Met Trp Leu Tyr 430 Va1 Leu Glu Asp 490 Gly Glu Arg Leu Ala 570 Ile Ala Leu Phe Gly 650 Thr Gin Tyr Glu Arg 730 Ala Leu Leu Gin Glu 475 Gin Lys Ala Glu Va1 555 Thr Pro Glu Arg Gin 635 Va1 Va1 Glu Phe Glu 715 Tyr Glu Met Ala Ala 460 Va1 Leu Thr Leu Leu 540 His Ala Val Ala Va1 620 Glu Pro Asn Leu Gin 700 Gly Va1 Arg Lys His 445 Leu Phe Glu Gin Arg 525 Thr Pro Thr Arg Gly 605 Leu Gly Pro Phe Ala 685 Ser Arg Pro Met Leu 765 Met Ser Arg Arg Lys 510 Glu Lys Arg Gly Thr 590 Trp Ala Lys Glu Gly 670 Ile Phe Lys Asp Ala 750 Ala Glu Leu Leu Val 495 Thr Ala Leu Thr Arg 575 Pro Ala His Asp Ala 655 Va1 Pro Pro Arg Leu 735 Phe Met Ala Glu Ala 480 Leu Gly His Lys Gly 560 Leu Leu Leu Leu Ile 640 Va1 Leu Tyr Lys Gly 720 Asn Asn Val r r r r

C

Glv Ale Aro Me+ Lall I.~ll r.ln 780 230- Val His Asp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg Ala Glu Glu 785 790 795 800 Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala 805 810 815 Val i'ro Leu Glu Val Glu Val Gly met Gly Glu Asp Trp Leu Ser Ala 820 825 830 Lys Gly INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 2502 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:?: ATGNNGGCGA TGCTTCCCCT CTTTGAGCCC AAAGGCCGGG TCCTCCTGGT GGACGGCCAC

CACCTGGCCT

GTGCAGGCGG

1NGGCGGTGN

GCCTACAAGG

AAGGAGCTGG

GACGTNCTGG

ACCGCCGACC

GGGTACCTCA

GTGGACTACC

GGGGAGAAGA

AACCTGGACC

ANGCTCTCCT

AAGNGGCGGG

AGCCTCCTCC

CCCCCGCCGG

GAGCTTCTGG

ANGGGCCTNA

GCCCTGAGGG

CTGGACCCCT

GAGGANGCGG

CTTGAGGGGG

GTCCTGGCCC

ACCGCACCTT

TCTACGGCTT

TCGTGGTCTT

CGGGCCGGGC

TGGACCTCCT

CCACCCTGGC

GCGACCTCTA

TCACCCCGGC

GGGCCCTGGC

CCGCCCNGAA

GGGTGAAGCC

GGGAGCTNTC

AGCCCGACCG

ACGAGTTCGG

AAGGGGCCTT

CCCTGGCCGC

GGGACCTNAA

AGGGCCTNGA

CCAACACCAC

GGGAGCGGGC

AGGAGAGGCT

ACATGGAGGC

CTTCGCCCTG

CGCCAAGAGC

TGACGCCAAG

CCCCACCCCG

GGGGCTTGCG

CAAGAAGGCG

CCAGCTCCTT

GTGGCTTTGG

GGGGGACCCC

GCTCCTCNAG

CGCCNTCCGG

CCAGGTGCGC

GGAGGGGCTT

CCTCCTGGAG

CGTGGGCT1T

CGCCAGGGAG

GGAGGTGCGG

CCTCNTGCCC

CCCCGAGGGG

CCTCCTNTCC

CCTTTGGCTT

CACGGGGGTN

AAGGGCCTCA

CTCCTCAAGG

GCCCCCTCCT

GAGGACTTTC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCA

GAGAAGTACG

TCCGACAACC

GAGTGGGGGA

GAGAAGATCC

ACCGACCTGC

AGGGCCTTTC

GGCCCCAAGG

GTCCTTTCCC

GGCCGGGTCC

GGNCTCCTCG

GGGGACGACC

GTGGCCCGGC

GAGAGGCTCT

TACCAGGAGG

CGGCTGGACG

CCACCAGCCG

CCCTGAAGGA

TCCGCCACGA

CCCGGCAGCT

TCCCCGGCTA

GGTACGAGGT

TCGCCGTCCT

GCCTGAGGCC

TCCCCGGGGT

GCCTGGAAAA

AGGCCCACAT

CCCTGGAGGT

TGGAGAGGCT

CCCTGGAGGA

GCCCCGAGCC

ACCGGGCACC

CCAAGGACCT

CCATGCTCCT

GCTACGGGGG

TCCNGAACCT

TGGAGAAGCC

TGGCCTACCT

GGGCGAACCG

GGACGGGGAC

GGCCTACGAG

CGCCCTCATC

CGAGGCGGAC

GCGCATCCTC

CCACCCCGAG

GGAGCAGTGG

CAAGGGCATC

CCTCCTCAAG

GGANGACCTG

GGACTTCGCC

GGAGTTTGGC

GGCCCCCTGG

CATGTGGGCC

AGACCCCT'rr

GGCCGTTTTG

CGCCTACCTC

GGAGTGGACG

NNNGCAGCGC

CCTTTCCCGG

CCAGGCCCTN

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 TCCCTGGAGG TGGCGGAGGA GATCCGCCGC CTCGAGGAGG AGGTCTTCCG CCTGGCCGGC 231 CACCCCTTCA ACCTCAACTC CTTCCCGCCA TCGGCAAGAC GAGGCCCTNC GNGAGGCCCA CTCCACACCC GCTTCAACCA AACCTGCAGA ACATCCCCGT GCCGAGGAGG GNTGGGTGTT GCCCACCTCT CCGGGGACGA ACCCAGACCG CCAGCTGGAT CGGGCGC-CCA AGACCATCAA CAGGAGCTTG CCATCCCCTA TTCCCCAAGG TGCGGGCCTG GTGGAGACCC TCTTCGGCCG GTGCGGGAGG CGGCGGAGCG CTCATGAAGC TGGCCATGGT CTCCTNCAGG TCCACGACGA GCCGCTT-TGG CCAAGGAGGT GAGGTGGGGA TGGGGGAGGA

CCGGGACCAC

GGAGAAGACN

CCCCATCGTG

GACGGCCACG

CCGCACCCCN

GGTGGCCCTG

GAACCTGATC

GTTCGGCGTC

CTTCGGGGTC

CGAGGAGGCG

GATTGAGAAG

CCGGCGCTAC

CATGGCCTTC

GAAGCTCTTC

GCTGGTCCTC

CATGGAGGGG

CTGGCTCTCC

CTGGAAAGGG

GGCAAGCGCT

GAGAAGATCC

CCNGNCCTCG

GCCACGGGCA

CTGGGCCAGA

GACTATAGCC

CGGGTCTTCC

CCCCCGGAGG

CTCTACGGCA

GTGGCCTTCA

ACCCTGGAGG

GTGCCCGACC

AACATGCCCG

CCCCGGCTNC

GAGGCCCCCA

GTCTATCCCC

GCCAAGGAGT

TGCTCTTTGA

CCACCAGCGC

TGCAGTACCG

TCCACCCCAG

GGCTTAGTAG

GGATCCGCCG

AGATAGAGCr

AGGAGGGGAG

CCGTGGACCC

TGTCCGCCCA

TTGAGCGCTA

AGGGCAGGAG

TCAACGCCCG

TCCAGGGCAC

AGGAAATGGG

AAGAGCGGGC

TGGCCGTGCC

AG

CGAGCTNGGG

CGCCGTGCTG

GGAGCTCACC

GACGGGCCGC

CTCCGACCCC

GGCCTTCGTG

CCGGGTCCTG

GGACATCCAC

CCTGATGCGC

CCGCCTCTCC

CTTCCAGAGC

GCGGGGGTAC

GGTGAAGAGC

CGCCGCCGAC

GGCCAGGATG

GGAGGNGGTG

CCTGGAGGTG

1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2502 INFORMATION FOR SEQ ID NO:8: Wa SEQUENCE CHARACTERISTICS: LENGTH: 833 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Xaa Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu ft Val Asp Gly His His Leu Ala Tyr Leu Thr Thr Ser Arg Gly Glu Pro 40 Lys Ser Leu Leu Lys Ala Leu Lys Arg Thr Phe 25 Val Gin Ala Glu Asp Gly Phe Ala Leu Lys Gly Val Tyr Gly Phe Ala Ala Val Xaa Val 55 Phe Asp Ala Lys Ala Pro Ser Phe Arg His 75 Ala Tyr Glu Tyr Lys Ala Gly Arg 85 Ala Leu Ile Lys Glu 100 Ala Pro Thr Leu Val Asp Pro Glu Asp S90 Phe Pro Arg Gln Leu Leu Glu Leu Leu Gly Leu Xaa Arg 105 110 232 Val Pro Gly Tyr Giu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Giu Val Arg Ile Leu Thr Ala Asp Arg Asp 130 135 140 Leu Ty i Jn Leu Leu Ser Asp Arg Ile Ala Val Leu His Pro Giu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Giu Gin Trp Val Asp Tyr Arg Ala Leu Xaa Gly Asp Pro Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Giy Ile Gly Glu Lys Thr Ala Xaa Lys Leu Leu 195 200 205 Xaa Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val 210 215 220 Lys Pro Xaa Xaa Arg Giu Lys Ile Xaa Ala His Met Giu Asp Leu Xaa 225 230 235 240 Leu Ser Xaa Xaa Leu Ser Xaa Val Arg Thr Asp Leu Pro Leu Giu Val 245 250 255 Asp Phe Ala Xaa Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala Phe 260 265 270 Leu Giu Arg Leu Glu Phe Gly Ser Leu Leu His Giu Phe Gly Leu Leu 275 280 285 Giu Xaa Pro Lys Ala Leu Glu Giu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Xaa Gly Arg Val His Arg Ala Xaa **325 -330 335 Asp Pro Leu Xaa Gly Leu Arg Asp Leu Lys Giu Val Arg Gly Leu Leu *340 345 350 Ala Lys Asp Leu Ala Val Leu Ala Leu Arg Giu Gly Leu Asp Leu Xaa *355 360 365 *Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Giu Trp Thr Glu 385 390 395 400 *Asp Ala Gly Giu Arg Ala Leu Leu Ser Glu Arg Leu Phe Xaa Asn Leu 405 410 415 Xaa Xaa Arg Leu Glu Gly Glu Giu Arg Leu Leu Trp Leu Tyr Xaa Glu 420 425 430 Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 ***Val Arg Leu Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Glu Val Ala *450 455 460 *Giu Glu Ile Arg Arg Leu Glu Giu Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 233 Pro Phe Asn Leu Asn 485 Ser Arg Asp Gin Leu Glu Arg Val Leu Phe Asp 490 495 Glu Val1 Tyr 545 H i s Ser Arg Le u Asp 625 Gin Leu Met Ala Ala 705 Glu Val1 Val1 Phe Asp 785 Al a Leu Xaa Leu 500 Ala Ile Pro Phe Asn 580 Arg Ser Leu Ser Arg 660 His Phe Glu Phe Val 740 Thr Leu Vai Lys Glu 820 234 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHAR~ACTERISTICS: LENGTH: 1647 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ATGAATTCGG

CACCACCTGG

CCGGTGCAGG

GACGCGGTGA

GGGTACAAGG

AAGGAGCTGG

GACGTCCTGG

ACCGCCGACA

GGGTACCTCA

GCCGACTACC

GGGGAGAAGA

AACCTGGACC

AAGCTCTCCT

AAAAGGCGGG

AGCCTCCTCC

CCCCCGCCGG

GATCTTCTGG

AAAGCCCTCA

GCCCTGAGGG

CTGGACCCTT

GAGGAGGCGG

CTTGAGGGGG

GTCCTGGCCC

TCCCTGGAGG

CACCCCTTCA

CTTCCCGCCA

GAGGCCCTCC

ACTGGCCGTC

GGATGC7GCC

CCTACCGCAC

CGGTCTACGG

TCGTGGTCTT

CGGGCCGGGC

TGGACCTCCT

CCAGCCTGGC

AAGACCTTTA

TCACCCCGGC

GGGCCCTGAC

CGGCGAGGAA

GGCTGAAGCC

GGGACCTGGC

AGCCCGACCG

ACGAGTTCGG

AAGGGGCCT'

CCCTGGCCGC

GGGACCTGAA

AAGGCCTTGG

CCAACACCAC

GGGAGCGGGC

AGGAGAGGCT

ACATGGAGGC

TGGCCGGGGA

ACCTCAACTC

TCGGCAAGAC

GCGAGGCCCA

GTTTTACAAC

CCTCTTTGAG CCCAAGGGCC

CTTCCACGCC

CTTCGCCAAG

TGACGCCAAG

CCCCACGCCG

GGGGCTGGCG

CAAGAAGGCG

CCAGCTCCTT

CTGGCTTTGG

CGGGGACGAG

GCTTCTGGAG

CGCCATCCGG

CAAGGTGCGC

GGAGAGGCTT

CCTTCTGGAA

CGTGGGCTTT

CGCCAGGGGG

GGAGGCGCGG

CCTCCCGCCC

CCCCGAGGGG

CGCCCTTTCC

CCTT TGGCTT

CACGGGGGTG

GATCGCCCGC

CCGGGACCAG

GGAGAAGACC

CCCCATCGTG

GTCGTGA

CTGAAGGGCC

AGCCTCCTCA

GCCCCCTCCT

GAGGACTTTC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCA

GAAAAGTACG

TCCGACAACC

GAGTGGGGGA

GAGAAGATCC

ACCGACCTGC

AGGGCCTTTC

AGCCCCAAGG

GTGCTTTCCC

GGCCGGGTCC

GGGCTTCTCG

GGCGACGACC

GTGGCCCGGC

GAGAGGCTCT

TACCGGGAGG

CGCCTGGACG

CTCGAGGCCG

CTGGAAAGGG

GGCAAGCGCT

GAGAAGATCC

GGGTCCTCCT

TCACCACCAG

AGGCCCTCAA

TCCGCCACGA

CCCGGCAACT

TCCCGGGCTA

GCTACGAGGT

TCCACGTCCT

GCCTGAGGCC

TTCCCGGGGT

GCCTGGAAGC

TGGCCCACAT

CCCTGGAGGT

TGGAGAGGCT

CCCTGGAGGA

GCAAGGAGCC

ACCGGGCCCC

CCAAAGACCT

CCATGCTCCT

GCTACGGCGG

TCGCCAACCT

TGGAGAGGCC

TGGCCTATCT

AGGTC7 TCCG

TCCTCTTTGA

CCACCAGCGC

TGCAGGCATG

GGTGGACGGC

CCGGGGGGAG

GGAGGACGGG

GGCCTACGGG

CGCCCTCATC

CGAGGCGGAC

CCGCATCCTC

CCACCCCGAG

CGACCAGTGG

CAAGGGCATC

CCTCCTCAAG

GGACGATCTG

GGACTTCGCC

TGAGTTTGGC

GGCCCCCTGG

CATGTGGGCC

CGAGCCTTAT

GAGCGTTCTG

CGCCTACCTC

GGAGTGGACG

GTGGGGGAGG

CCTTTCCGCT

CAGGGCCTTG

CCTGGCCGGC

CGAGCTAGGG

CGCCGTCCTG

CAAGCTTGGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1647

S

S

S S S S 55 235 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2088 base pairs TYPE: nucleic acid STRAI1-DE'4DNESS: double TOCILOC. lilieza (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATGAATTCGG GGATGCTGCC CCTCTTTGAG

C

C

a.

CACCACCTGG

CCGGTGCAGG

GACGCGGTGA

GGGTACAAGG

AAGGAGCTGG

GACGTCCTGG

ACCGCCGACA

GGGTACCTCA

GCCGACTACC

GGGGAGAAGA

AACCTGGACC

AAGCTCTCCT

AAAAGGCGGG

AGCCTCCTCC

CCCCCGCCGG

GATCTTCTGG

AAAGCCCTCA

GCCCTGAGGG

CTGGACCCTT

GAGGAGGCGG

CTTGAGGGGG

GTCCTGGCCC

TCCCTGGAGG

CACCCCTTCA

CTTCCCGCCA

GAGGCCCTCC

AAGCTGAAGA

CTCCACACCC

CCTACCGCAC

CGGTCTACGG

TCGTGGTCTT

CGGGCCGGGC

TGGACCTCCT

CCAGCCTGGC

AAGACCTTTA

TCACCCCGGC

GGGCCCTGAC

CGGCGAGGAA

GGCTGAAGCC

GGGACCTGGC

AGCCCGACCG

ACGAGTTCGG

AAGGGGCCTT

C CCTG GCCG C

GGGACCTGAA

AAGGCCTTGG

CCAACACCAC

GGGAGCGGGC

AGGAGAGGCT

ACATGGAGGC

TGGCCGGGGA

ACCTCAACTC

TCGGCAAGAC

GCGAGGCCCA

GCACCTACAT

GCTTCAACCA

CTTCCACGCC

CTTCGCCAAG

TGACGCCAAG

CCCCACGCCG

GGGGCTGGCG

CA.AGAAGGCG

CCAGCTCCTT

CTGGCTTTGG

CGGGGACGAG

GCTTCTGGAG

CGCCATCCGG

CAAGGTGCGC

GGAGAGGCTT

CCTTCTGGAA

CGTGGGCTTT

CGCCAGGGGG

GGAGGCGCGG

CCTCCCGCCC

CCCCGAGGGG

CGCCCTTTCC

CCTTrGGCTT

CACGGGGGTG

GATCGCCCGC

CCGGGACCAG

GGAGAAGACC

CCCCATCGTG

TGACCCCTTG

GACGGCCACG

CCCAAGGGCC

CTGAAGGGCC

AGCCTCCTCA

GCCCCCTCCT

GAGGACTTTC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCA

GAAAAGTACG

TCCGACAACC

GAGTGGGGGA

GAGAAGATCC

ACCGACCTGC

AGGGCCTTTC

AGCCCCAAGG

GTGCTTTCCC

GGCCGGGTCC

GGGCTTCTCG

GGCGACGACC

GTGGCCCGGC

GAGAGGCTCT

TACCGGGAGG

CGCCTGGACG

CTCGAGGCCG

CTGGAAAGGG

GGCA.AGCGCT

GAGAAGATCC

CCGGACCTCA

GCCACGGGCA

GGGTCCTCCT

TCACCACCAG

AGGCCCTCAA

TCCGCCACGA

CCCGGCAACT

TCCCGGGCTA

GCTACGAGGT

TCCACGTCCT

GCCTGAGGCC

TTCCCGGGGT

GCCTGGAAGC

TGGCCCACAT

CCCTGGAGGT

TGGAGAGGCT

CCCTGGAGGA

GCAAGGAGCC

ACCGGGCCCC

CCAAAGACCT

CCATGCTCCT

GCTACGGCGG

TCGCCAACCT

TGGAGAGGCC

TGGCCTATCT

AGGTCTTCCG

TCCTCTTTGA

CCACCAGCGC

TGCAGTACCG

TCCACCCCAG

GGCTAAGTAG

GGTGGACGGC

CCGGGGGGAG

GGAGGACGGG

GGCCTACGGG

CGCCCTCATC

CGAGGCGGAC

CCGCATCCTC

CCACCCCGAG

CGACCAGTGG

CAAGGGCATC

CCTCCTCAAG

GGACGATCTG

GGACTTCGCC

TGAGTTTGGC

GGCCCCCTGG

CATGTGGGCC

CGAGCCTTAT

GAGCGTTCTG

CGCCTACCTC

GGAGTGGACG

GTGGGGGAGG

CCTTTCCGCT

CAGGGCCTTG

CCTGGCCGGC

CGAGCTAGGG

CGCCGTCCTG

GGAGCTCACC

GACGGGCCGC

CTCCGATCCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 236 AACCTCCAGA ACATCCCCGT CCGCACCCCG CTTGGGCAGA GGATCCGCCG GGCCTTCATC GCCGAGGAGG GGTGGCTATT GGTGGCCCTG GACTATAGCC AGATAGAGCT CAGGGTGCTG GCCCACCTCT CCGGCGACGA GAACCTGATC CGGGTCTTCC AGGAGGGGCG GGACATCCAC ACGGAGACCG CCAGCTGGAT GTTCGGCGTC CCCCGGGAGG CCGTGGACCC CCTGATGCGC CGGGCGGCCA AGACCATCAA CTTCGGGGTC CTCTACGGCA TGTCGGCCCA CCGCCTCTCC CAGGAGCTAG CTAGCCATCC CTTACGAGGA GGCCCAGGCC TTCATTGA INFORMATION FOR SEQ ID NO:l1: SEQUENCE CHARACTERISTICS: LENGTH: 962 base pairs TYPE: nucleic acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: 1800 1860 1920 1980 2040 2088 ATGAATTCGG GGATGCTGCC CCTCTTTGAG CCCAAGGGCC GGGTCCTCCT 55 S S 5

S*

S

SSS S S S. 55

S

5 S. S 55

CACCACCTGG

CCGGTGCAGG

GACGCGGTGA

GGGTACAAGG

AAGGAGCTGG

GACGTCCTGG

ACCGCCGACA

GGGTACCTCA

GCCGACTACC

GGGGAGA.AGA

AACCTGGACC

AAGCTCTCCT

AAAAGGCGGG

AGCCTCCTCC

TGGCCGTGCC

CCTACCGCAC

CGGTCTACGG

TCGTGGTCTT

CGGGCCGGGC

TGGACCTCCT

CCAGCCTGGC

AAGACCTTTA

TCACCCCGGC

GGGCCCTGAC

CGGCGAGGAA

GGCTGAAGCC

GGGACCTGGC

AGCCCGACCG

ACGAGTTCGG

CCTGGAGGTG

CTTCCACGCC

CTTCGCCAAG

TGACGCCAAG

CCCCACGCCG

GGGGCTGGCG

CAAGAAGGCG

CCAGCTTCTT

CTGGCfl'GG

CGGGGACGAG

GCTTCTGGAG

CGCCATCCGG

CAAGGTGCGC

GGAGAGGCTT

CTGAAGGGCC

AGCCTCCTCA

GCCCCCTCCT

GAGGACTTTC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCA

GAAAAGTACG

TCCGACAACC

GAGTGGGGGA

GAGAAGATCC

ACCGACCTGC

AGGGCCTTTC

TCACCACCAG

AGGCCCTCAA

TCCGCCACGA

CCCGGCAACT

TCCCGGGCTA

GCTACGAGGT

TCCACGTCCT

GCCTGAGGCC

TTCCCGGGGT

GCCTGGAAGC

TGGCCCACAT

CCCTGGAGGT

TGGAGAGGCT

CATGGAGGGG

CTGGCTCTCC

GGTGGACGGC

CCGGGGGGAG

GGAGGACGGG

GGCCTACGGG

CGCCCTCATC

CGAGGCGGAC

CCGCATCCTC

CCACCCCGAG

CGACCAGTGG

CAAGGGCATC

CCTCCTCAAG

GGACGATCTG

GGAC TTCGCC

TG.AGTTTGGC

GTGTATC CCC

GCCAAGGAGT

CCTTCTGGA.A AGCCCCAAGT GAGGTGGGGA TAGGGGAGGA INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 1600 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 237 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATGGAATTCG GGGATGCTGC CCCTCTTTGA

GCCCAAGGGC

CCACCACCTG GCCTACCGCA CCTTCCACGC GCTGCA, GCGCTACU GCTTCGCCA; GGACGCGGTG ATCGTGGTCT T'rGACGCCA; GGGGTACAAG GCGGGCCGGG CCCCCACGCC CAAGGAGCTG GTGGACCTCC TGGGGCTGGC CGACGTCCTG C-CCAGCCTGG CCAAGAAGGC CACCGCCGAC AAAGACCTTT ACCAGCTCCT GGGGTACCTC ATCACCCCGG

CCTGGCTTTG

GGCCGACTAC CGGGCCCTGA

CCGGGGACGA

CGGGGAGAAG ACGGCGAGGA

AGCTTCTGGA

GAACCTGGAC CGGCTGAAGC CCGCCATCCG GAAGCTC'.!CC TGGGACCTGG CCAAGGTGCG CAAAAGGCGG GAGCCCGACC GGGAGAGGCT CAGCCTCCTC CACGAGTTCG GCCTTCTGGA CGAGGAGGGG TGGCTATTGG TGGCCCTGGA CCACCTCTCC GGCGACGAGA ACCTGATCCG GGAGACCGCC AGCTGGATGT TCGGCGTCCC GGCGGCCAAG ACCATCAACT TCGGGGTCCT GGAGCTAGCC ATCCCTTACG AGGAGGCCCA CCCCAAGGTG CGGGCCTGGA TTGAGAAGAC GGAGACCCTC TTCGGCCGCC GCCGCTACGT GCGGGAGGCG GCCGAGCGCA TGGCC'ITCAA CATGAAGCTG GCTATGGTGA AGCTCTTCCC CCTTCAGGTC CACGACGAGC TGGTCCTCGA.

CCGGCTGGCC AAGGAGGTCA TGGAGGGGGT GGTGGGGATA GGGGAGGACT GGCTCTCCGC

CCTGAAGGGC

GAGCCTCCTC

LGGCCCCCTCC

GGAGGACTT

GCGCCTCGAG

*GGAAAAGGAG

*TTCCGACCGC

GGAAAAGTAC

GTCCGACAAC

GGAGTGGGGG

GGAGAAGATC

CACCGACCTG

TAGGGCCTT'r

AAGCCCCAAG

CTATAGCCAG

GGTCTT1CCAG

CCGGGAGGCC

CTACGGCATG

GGCCTTCATT

CCTGGAGGAG

GCCAGACCTA

CATGCCCGTC

CAGGCTGGAG

GGCCCCAAAA

GTATCCCCTG

CAAGGAGTGA

CGGGTCCTCC TGGTGGACGG CTCACCACCA GCCGGGGGGA

*AAGGCCCTCA

TTCCGCCACG

CCCCGGCAAC

GTCCCGGGCT

GGCTACGAGG

ATCCACGTCC

GGCCTGAGGC

CTTCCCGGGG

AGCCTGGAAG

CTGGCCCACA

CCCCTGGAGG

CTGGAGAGGC

ATCCGCCCGGG

ATAGAGCTCA

GAGGGGCGGG

GTGGACCCCC

TCGGCCCACC

GAGCGCTACT

GGCAGGAGGC

GAGGCCCGGG

CGGGGCACCG

GAAATGGGGG

GAGAGGGCGG

GCCGTGCCCC

AGGAGGACGG

AGGCCTACGG

TCGCCCTCAT

ACGAGGCGGA

TCCGCATCCT

TCCACCCCGA

CCGACCAGTG

TCAAGGGCAT

CCCTCCTCAA

TGGACGATCT

TGGACTTCGC

TTGAGTTTGG

CCTTCATCGC

GGGTGCTGGC

ACATCCACAC

TGATGCGCCG

GCCTCTCCCA

TTCAGAGCTT

GGGGGTACGT

TGAAGAGCGT

CCGCCGACCT

CCAGGATGCT

AGGCCGTGGC

TGGAGGTGGA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1600 C

S

INFORMATION FOR SEQ ID NO:13: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CACGAATTCG GGGATGCTGC CCCTCTTTGA GCCCAA C. CC C S

C.

C. C C CC C CC 238 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTGAGATCTA TCACTCCTTG GCGGAGAGCC AGTC 34 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 91 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID TAATACGACT CACTATAGGG AGACCGGAAT TCGAGCTCGC CCGGGCGAGC TCGAATTCCG TGTATTCTAT AGTGTCACCT AAATCGAATT C 91 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TAATACGACT CACTATAGGG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAATTCGATT TAGGTGACAC TATAGAA 27 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) e 239 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GTAATCATGG TCATAGCTGG TAGCTTGCTA C INFORMATION FOR SEQ ID NO:19: ()SEQUENCE CIIARA-TERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGATCCTCTA GAGTCGACCT GCAGGCATGC CTACCTTGGTr AG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRAkNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCTCTA GAGTCGACCT GCAGGCATGC INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 2502 base pairs TYPE: nucleic acid STR.ANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 0t 0:0 0:6 *see 0 000 6000* 0@ 0 @00

ATGAATTCGG

CACCACCTGG

CCGGTGCAGG

GACGCGGTGA

GGGTACA-AGG

AAGGAGCTGG

GACGTCCTGG

ACCGCCGACA

GGGTACCTCA

GCCGACTACC

GGGGAGAAGA

AACCTGGACC

AAGCTCTCCT

GGATGCTGCC

CCTACCGCAC

CGGTCTACGG

TCGTGGTCTT

CGGGCCGGGC

TGGACCTCCT

CCAGCCTGGC

AAGACCTTTA

TCACCCCGGC

GGGCCCTGAC

CGGCGAGGAA

GGCTGAAGCC

GGGACCTGGC

CCTCTTTGAG

CTTCCACGCC

CTTCGCCAAG

TGACGCCAAG

CCCCACGCCG

GGGGCTGGCG

CAAGAAGGCG

CCAGCTCCTT

CTGGCTTTGG

CGGGGACGAG

GCTTCTGGAG

CGCCATCCGG

CAAGGTGCGC

CCCAAGGGCC

CTGAAGGGCC

AGCCTCCTCA

GCCCCCTCCT

GAGGACTTTC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCA

GAAAAGTACG

TCCGACAACC

GAGTGGGGGA

GAGAAGATCC

ACCGACCTGC

GGGTCCTCCT

TCACCACCAG

AGGCCCTCAA

TCCGCCACGA

CCCGGCAACT

TCCCGGGCTA

GCTACGAGGT

TCCACGTCCT

GCCTGAGGCC

TTCCCGGGGT

GCCTGGA.AGC

TGGCCCACAT

CCCTGGAGGT

GGTGGACGGC

CCGGGGGGAG

GGAGGACGGG

GGCCTACGGG

CGCCCTCATC

CGAGGCGGAC

CCGCATC CT C

CCACCCCGAG

CGACCAGTGG

CAAGGGCATC

CCTCCTCAAG

GGACGATCTG

GGACTTCGCC

240

AAAAGGCGGG

AGCCTCCTCC

CCCCCGCCGG

AAAGCCCTCA

GCCCTGAGGG

CTGGACCCTT

GAGGAGGCGG

CTTGAGGGGG

GTCCTGG CCC

TCCCTGGAGG

CACCCCTTCA

CTTCCCGCCA

GAGGCCCTCC

AAGCTGAAGA

CTCCACACCC

AACCTCCAGA

GCCGAGGAGG

GCCCACCTCT

ACGGAGACCG

CGGGCGGCCA

CAGGAGCTAG

TTCCCCAAGG

GTGGAGACCC

GTGCGGGAGG

CTCATGAAGC

CTCCTTCAGG

GCCCGGCTGG

AGCCCGACCG

ACGAGTTCGG

AAGGGGCCTT

GGGACCTGAA

AAGGCCTTGG

CCAACACCAC

GGGAGCGGGC

AGGAGAGGCT

ACATGGAGGC

TGGCCGGGGA

AC CTCAACT C

TCGGCAAGAC

GCGAGGCCCA

GCACCTACAT

GCTTCAACCA

ACATCCCCGT

GGTGGCTATT

CCGGCGACGA

CCAGCTGGAT

AGACCATCA-A

CCATCCCTTA

TGCGGGCCTG

TCTTCGGCCG

CGGCCGAGCG

TGGCTATGGT

TCCACGACGA

CCAAGGAGGT

GGAGAGGCTT AGGGCCTTTC CCTTCTGGAA AGCCCCAAGG

CGTGGGCT

GGAGGCGCGG

CCTCCCGCCC

CCCCGAGGGG

CGCCCTTTCC

CCTTTGGCTT

CACGGGGGTG

GATCGCCCGC

CCGGGACCAG

GGAGAAGACC

CCCCATCGTG

TGACCCCTTG

GACGGCCACG

CCGCACCCCG

GGTGGCCCTG

GAACCTGATC

GTTCGGCGTC

CTTCGGGGTC

CGAGGAGGCC

GATTGAGAAG

CCGCCGCTAC

CATGGCCTTC

GAAGCTCTTC

GCTGGTCCTC

CATGGAGGGG

GTGCTTTCCC

GGCGG~ i CC

GGGCTTCTCG

GGCGACGACC

GTGGCCCGGC

GAGAGGCTCT

TACCGGGAGG

CGCCTGGACG

CTCGAGGCCG

CTGGAAAGGG

GGCAAGCGCT

GAGAAGATCC

CCGGACCTCA

GCCACGGGCA

CTTGGGCAGA

GACTATAGCC

CGGGTCTTCC

CCCCGGGAGG

CTCTACGGCA

CAGGCCTTCA

ACCCTGGAGG

GTGCCAGACC

AACATGCCCG

CCCAGGCTGG

GAGGCCCCAA

GTGTATCCCC

GCCAAGGAGT

TGGAGAGGCT

CCCTGGAGGA

GCAAGGAGCC

AUCGGGCCCC

CCAA.AGACCT

CCATGCTCCT

GCTACGGCGG

TCGCCAACCT

TGGAGAGGCC

TGGCCTATCT

AGGTCTTCCG

TCCTCTTTGA

CCACCAGCGC

TGCAGTACCG

TCCACCCCAG

GGCTAAGTAG

GGATCCGCCG

AGATAGAGCT

AGGAGGGGCG

CCGTGGACCC

TGTCGGCCCA

TTGAGCGCTA

AGGGCAGGAG

TAGAGGCCCG

TCCGGGGCAC

AGGAAATGGG

AAGAGAGGGC

TGGCCGTGCC

GA

TGAGTTTGGC

GGCCCCCTGG

CATGTGGGCC

CGAGC=rAT

GAGCGTTCTG

CGCCTACCTC

GGAGTGGACG

GTGGGGGAGG

CCTTTCCGCT

CAGGGCCTTG

CCTGGCCGGC

CGAGCTAGGG

CGCCGTCCTG

GGAGCTCACC

GACGGGCCGC

CTCCGATCCC

GGCCTTCATC

CAGGGTGCTG

GGACATCCAC

CCTGATGCGC

CCGCCTCTCC

CTTTCAGAGC

GCGGGGGTAC

GGTGAAGAGC

CGCCGCCGAC

GGCCAGGATG

GGAGGCCGTG

CCTGGAGGTG

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2502 GAGGTGGGGA TAGGGGAGGA CTGGCTCTCC INFORMATION FOR SEQ ID NO:22: SEQUENCE CHRPACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 241 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GATTTAGGTG ACACTATAG INFORMATION FOR SEQ ID NO:23: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGGACGAACA AGCGAGACAG CGACACAGGT ACCACATGGT ACAAGAGGCA AGAGAGACGA CACAGCAGAA AC INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GTTTCTGCTG TGTCGTCTCT CTTGCCTCTT GTACCATGTG GTACCTGTGT CGCTGTCTCG CTTGTTCGTC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GACGAACAAG CGAGACAGCG INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GTTTCTGCTG TGTCGTCTCT CTTG 24 *o e oo -242- INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 46 base pairs TYPE: nucleic acid STRANDEDNESS: single (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CCTCTTGTAC CATGTGGTAC CTGTGTCGCT GTCTCGCTTG TTCGTC INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ACACAGGTAC CACATGGTAC AAGAGGCAAG AGAGACGACA CAGCAGAAAC INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Met Ala Ser Met Thr Gly Gly Gin Gin Met Gly Arg Ile Asn Ser 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 969 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGGATCA ATTCGGGGAT TTTGAGCCCA AGGGCCGGGT CCTCCTGGTG GACGGCCACC ACCTGGCCTA CACGCCCTGA AGGGCCTCAC CACCAGCCGG GGGGAGCCGG TGCAGGCGGT GCCAAGAGCC TCCTCAAGGC CCTCAAGGAG GACGGGGACG CGGTGATCGT GCCAAGGCCC CCTCCTTCCG CCACGAGGCC TACGGGGGGT ACAAGGCGGG ACGCCGGAGG ACTTTCCCCG GCAACTCGCC CTCATCAAGG AGCTGGTGGA CTGGCGCGCC TCGAGGTCCC GGGCTACGAG GCGGACGACG TCCTGGCCAG AAGGCGGAAA AGGAGGGCTA CGAGGTCCGC ATCCTCACCG CCGACAAAGA

GCTGCCCCTC

CCGCACCTTC

CTACGGCTTC

GGTCTTTGAC

CCGGGCCCCC

CCTCCTGGGG

CCTGGCCAAG

CCTTTACCAG

243 CTTCTTTCCG ACCGCATCCA CGTCCTCCAC CCCGAGGGGT ACCTCATCAC CTTTGGGAAA AGTACGGCCT GAGGCCCGAC CAGTGGGCCG ACTACCGGGC GACGAGTCCG ACAACCTTCC CGGGGTCAAG GGCATCGGGG AGAAGACGGC CTGGAGGAGT GGGGGAGCCT GGAAnrrr-CtC TC AACC TGGACCGGCT ATCCGGGAGA AGATCCTGGC CCACATGGAC GATCTGAAGC TCTCCTGGGA GTGCGCACCG ACCTGCCCCT GGAGGTGGAC TTCGCCAAAA GGCGGGAGCC AGGCTTAGGG CCTTTCTGGA GAGGCTTGAG TTTGGCAGCC TCCTCCACGA CTGGAAAGCC CCAAGTCATG GAGGGGGTGT ATCCCCTGGC CGTGCCCCTG

TGGGGATAG

INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 948 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECUL~E TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

CCCGGCCTGG

CCTGACCGGG

GAGGAAGCTT

GAAGCCCGCC

CCTGGCCAAG

CGACCGGGAG

GTTCGGCCTT

GAGGTGGAGG

ATGGCTAGCA TGACTGGTGG

TTTGAGCCCA

CACGCCCTGA

GCCAAGAGCC

GCCAAGGCCC

ACGCCGGAGG

CTGGCGCGCC

AAGGCGGAAA

CTTCTTTCCG

CTTTGGGAAA.

GACGAGTCCG

CTGGAGGAGT

ATCCGGGAGA

GTGCGCACCG

AGGCTTAGGG

CTGGAAAGCC

AGGGCCGGGT

AGGGCCTCAC

TCCTCAAGGC

CCTCCTTCCG

ACTTTCCCCG

TCGAGGTCCC

AGGAGGGCTA

ACCGCATCCA

AGTACGGCCT

ACAACCTTCC

GGGGGAGCC'T

AGATCCTGGC

ACCTGCCCCT

CCTTTCTGGA

CCAAGGCCGC

ACAGCAAATG

CCTCCTGGTG

CACCAGCCGG

CCTCAAGGAG

CCACGAGGCC

GCAACTCGCC

GGGCTACGAG

CGAGGTCCGC

CGTCCTCCAC

GAGGCCCGAC

CGGGGTCAAG

GGAAGCCCTC

CCACATGGAC

GGAGGTGGAC

GAGGCTTGAG

ACTCGAGCAC

GACGGCCACC

GGGGAGCCGG

GACGGGGACG

TACGGGGGGT

CTCATCAAGG

GCGGACGACG

ATCCTCACCG

CCCGAGGGGT

CAGTGGGCCG

GGCATCGGGG

CTCAAGAACC

GATCTGAAGC

TTCGCCAAAA

TTTGGCAGCC

CACCACCACC

ACCTGGCCTA

TGCAGGCGGT

CGGTGATCGT

ACAAGGCGGG

AGCTGGTGGA

TCCTGGCCAG

CCGACAAAGA

ACCTCATCAC

ACTACCGGGC

AGAAGACGGC

TGGACCGGCT

TCTCCTGGGA

GGCGGGAGCC

TCCTCCACGA

ACCACTGA

CCGCACCTTC

CTACGGCTTC

GGTCTTTGAC

CCGGGCCCCC

CCTCCTGGGG

CCTGGCCAAG

CCTTTACCAG

CCCGGCCTGG

CCTGACCGGG

GAGGAAGCTT

GAAGCCCGCC

CCTGGCCAAG

CGACCGGGAG

GTTCGGCCTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 948 GGTCGGATCA ATTCGGGGAT GCTGCCCCTC INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 206 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear 244 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC CAGTGAATTG TAATACGACT CACTATACCG CGAATuCGAG CTCUUTACCC GGGGATCCTC TAGAGTCGAC CTGCAGGCAT 120 GCAAGCTTGA GTATTCTATA GTGTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG 180 TTTCCTGTGT GAAATTGTTA TCCGCT 206 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: TTCTGGGTTC TCTGCTCTCT GGTCGCTGTC TCGCTTGTTC GTC 43 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GCTGTCTCGC TTGTTCGTC 19 INFORMATION FOR SEQ ID SEQUENCE CKARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) S* (xi) SEQUENCE DESCRIPTION: SEQ ID GACGAACAAG CGAGACAGCG INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear I' (ii) MOLECULE TYPE: DNA (genomic) S* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TTCTGGGTTC TCTGCTCTCT GGTC 24 24 245 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGV: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GACGAACAAG CGAGACAGCG ACCAGAGAGC AGAGAACCCA GAA 43 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ACCAGAGAGC AGAGAACCCA GAA 23 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: AACAGCTATG ACCATGATTA C 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GTTCTCTGCT CTCTGGTCGC TGTCTCGCTT GTGAAACAAG CGAGACAGCG TGGTCTCTCG INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S. (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: CGAGAGACCA CGCTG *246 -246- INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: sinale TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TC 52 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AGAAAGGAAG GGAAGAAAGC GAAAGG 26 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GACGGGGAAA GCCGGCGAAC G 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GAAAGCCGGC GAACGTGGCG INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: SGGCGAACGTG GCGAGAAAGG A 21 -247 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOui: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GC 42 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CCTTTCGCTC TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GC 42 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: modified base LOCATION: 8 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /evidence= EXPERIMENTAL /modbase= OTHER /note= "The A residue at this position is 2'-O-methyladenosine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: AGAAAGGAAG GGAAGAAAGC GAAAGGT 27 S* INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCCGGCGAAC GTGGCGAGAA AGGA 24 24 -248- INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: sinlp.

TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: GGTTTTTCTT TGAGGTTTAG INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GCGACACTCC ACCATAGAT 19 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CTGTCTTCAC GCAGAAAGC 19 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GCACGGTCTA CGAGACCTC 19 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID TAATACGACT CACTATAGGG -249- .i INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 337 base pairs TYPE: nucleic acid STRANDEDNESS: not relevant I(D1 UTOPOLOGY: not relevant (ii) MOLECULE TYPE: RNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: GGGAAAGCUU GCAUGCCUGC AGGUCGACUC UAGAGGAUCU ACUAGUCAUA UGGAUUCUGU CUUCACGCAG AAAGCGUCUG GCCAUGGCGU UAGUAUGAGU GUCGUGCAGC CUCCAGGACC 120 CCCCCUCCCG GGAGAGGCAU AGUGGUCUGC GGAACCGGUG AGUACACCGG AAUUGCCAGG 180 ACGACCGGGU CCUUUCUUGG AUAAACCCGC UCAAUGCCUG GAGAUUUGGG CGUGCCCCCG 240 CAAGACUGCU AGCCGAGUAG UGUUGGGUCG CGAAAGGCCU UGUGGUACUG CCUGAUAGGG 300 UGCCUGCGAG UGCCCCGGGA GGUCUCGUAG ACCGUGC 337 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: misc feature LOCATION: 18 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /evidence= EXPERIMENTAL /note= "The N at this position indicates the presence of a fluorescein dye on an abasic linker." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CCGGTCGTCC TGGCAATNCC INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" S(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: GTTTATCCAA GAAAGGACCC GGTC 24 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 250 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CAGGGTGAAG GGAAGAAGAA AGCGAAAGGT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CAGGGGGAAG GGAAGAAGAA AGCGAAAGGT INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: modified base LOCATION: 1..2 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /evidence= EXPERIMENTAL /mod base= OTHER /note= "The T residues at positions 1 and 2 are amino modified T residues." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: TTCTTTTCAC CAGCGAGACG GG 22 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: ATTGGGCGCC AGGGTGGTTT TT 22 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 53 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CCCGTCTCGC TGGTGAAAAG AAAAACCACC CTGGCGCCCA ATACGCAAAC CGC 53 -251 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GAATTCGATT TAGGTGACAC TATAGAATAC A 31 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GC 42 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: GCCGGCGAAC GTGGCGAGAA AGGA 24 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CAGAAGGAAG GGAAGAAAGC GAAAGG 26 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: S*CAGGGGGAAG GGAAGAAAGC GAAAGG 26 252 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single STOPOLOGY ru liinear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CAGGGTACAG GGAAGAAAGC GAAAGG 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: modified base LOCATION: 24 OTHER INFORMATION: /mod base= OTHER /note= "The residue at this position is a dideoxycytidine." (xi) SEQUENCE DESCRIPTION: SEQ ID GCCGGCGAAC GTGGCGAGAA AGGC 24 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(27, OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a Cy3 amidite group." (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 28 OTHER INFORMATION: /mod base= OTHER /note= "The residue at this position is a dideoxycytidine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: AGAAAGGAAG GGAAGAAAGC GAAAGGNC 28 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S253 -253

I

(ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, (uj UTHER INFORMATION: /note= "The residue at this position is a spacer containing a fluorescein label." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(28, OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a Cy3 amidite group." (ix) FEATURE: NAME/KEY: modified base LOCATION: 29 OTHER INFORMATION: /mod base= OTHER /note= "The residue at this position is a dideoxycytidine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: NAGAAAGGAA GGGAAGAAAG CGAAAGGNC 29 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a spacer containing a fluorescein label." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: NAGAAAGGAA GGGAAGAAAG CGAAAGG 27 INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this o. position is a spacer bearing a Cy3 amidite group." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(28, OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a biotin group." -254 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: NAGAAAGGAA GGGAAGAAAG CGAAAGGNT 29 INFORMATION FOR SEQ ID (i SEQUENCE CHARiACTEr KTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID GGAAAGCCGG CGAACGTGGC GAGA 24 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GGAAAGCCGG CGAACGTGGC GAGAAA 26 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, o**o OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a Cy3 amidite group." (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 2..3 OTHER INFORMATION: /mod base= OTHER /note= "The residues at these positions have an amino group added." (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(24, OTHER INFORMATION: /note= "The residue at this position is a spacer containing a fluorescein label." o.o: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: NTTCCAGAGC CTAATTTGCC AGTNA *5* 255 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single

TOPOTOGY

v linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: modified base LOCATION: 1 OTHER INFORMATION: /mod base= OTHER /note= "The residue at this position has a 5' TET-label." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(23, OTHER INFORMATION: /note= "The residue at this position is a spacer containing a fluorescein label." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: TTCCAGAGCC TAATTTGCCA GTNA 24 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: CTTACCAACG CTAACGAGCG TCTTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(14, oo*" OTHER INFORMATION: /note= "The residue at this positions contain an abasic ribose." (xi) SEQUENCE DESCRIPTION: SEQ ID CGAGAGACCA CGCT 14 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -256- (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(14, OTht INFORMATION: /note= "The residue at this position contains an abasic ribose with a 3' phosphate group." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CGAGAGACCA CGCT 14 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(15, OTHER INFORMATION: /note= "The residue at this position contains a 3' phosphate group." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: CGAGAGACCA CGCTG INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: CCCGTCTCGC TGGTGAAAAG AAAAACCACC CTGGCGCCCA ATA 43 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(16, OTHER INFORMATION: /note= "The residue at this position is a 3-nitropyrrole." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TATTGGGCGC CAGGGNGGTT TTT 23 ag 257 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOG': linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(16, OTHER INFORMATION: /note= "The residue at this position is a 3-nitropyrrole group." (xi) SEQUENCE DESCRIPTION: SEQ ID TATTGGGCGC CATGGNGGTT TTT 23 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: TATTGGGCGC CATGGTGGTT TTT 23 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(10, OTHER INFORMATION: /note= "The residue at this position is a (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(16, OTHER INFORMATION: /note= "The residue at this •position is a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: TATTGGGCGN CAGGGNGGTT TTT 23 o INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -258 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(10, OTHER INFORMATION: /note= "The position is a (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(16, OTHER INFORMATION: /note= "The position is a residue at this residue at this (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: TATTGGGCGN CATGGNGGTT TTT INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 9 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a Cy3 amidite label." (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 2..3 OTHER INFORMATION: /mod base= OTHER /note= "The residues at these-positions added." have an amino group .0 9 0 4...4 eS 6 9 0 0O 0 0 60 S

S

0e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:

NTTCACCAG

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyguanosine (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(2, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine -259 (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(3, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(4, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'--(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(5..6, OTHER INFORMATION: /note= "The residues at these positions are 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(7..8, OTHER INFORMATION: /note= "The residues at these positions are 2'deoxyguancsine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(9, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (xi) SEQUENCE DESCRIPTION: SEQ ID GCTCAAGGCA CTCTTGCCTA CGA 23 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid V0 DESCRIPTION: /desc "DNA" 0* (ix) FEATURE: 0 0(A) NAME/KEY: miscdifference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this o* position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." #fe FEATURE: NAME/KEY: miscdifference LOCATION: replace(2, OTHER INFORMATION: /note= "The residue at this 04: position is a 2'deoxythymidine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: 66900(A) NAME/KEY: misc difference LOCATION: replace(3..4, OTHER INFORMATION: /note= "The residues at these 6*00: positions are a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: 0 NAME/KEY: misc difference LOCATION: replace(5..6, OTHER INFORMATION: /note= "The residues at these :0.0*0 positions are a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." 0* 0 *0 6 0 -260- (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(7, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." NAME/KEY: misc difference LOCATION: replace(8, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(9, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate).

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: CTCCAACTAC CACAAGTTTA TATTCAG 27 INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 56 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(2, CD) OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(3, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyguanosine 5'-O-(1-Thiomonophosphate)."

FEATURE:

NAME/KEY: misc difference LOCATION: replace(4..5, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-0-(1-Thiomonophosphate).

(ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(6, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(7, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." 261 (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(8, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) E.IP~ NAME/KEY: misc difference LOCATION: replace(9..10, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: CTGAATATAA ACTTGTGGTA GTTGGAGCTG GTGACGTAGG CAAGAGTGCC TTGACG 56 INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 56 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(1, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference B) LOCATION: replace(2, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)."

FEATURE:

S(A) NAME/KEY: misc difference LOCATION: replace(3, OTHER INFORMATION: /note= "The residue at this ***position is a 2'deoxyguanosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(4..5, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-0-(1-Thiomonophosphate)."

FEATURE:

NAME/KEY: misc difference LOCATION: replace(6, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(7, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(8, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-0-(1-Thiomonophosphate)." 262 (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(9..10, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine (vi) E CRIPTION: SEQ ID VU:93: CTGAATATAA ACTTGTGGTA GTTGGAGCTG GTGCCGTAGG CAAGAGTGCC TTGACG 56 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position has a TET label." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: CCGGTCGTCC TGGCAA 16 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc_difference S* LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position has a TET label." (xi) SEQUENCE DESCRIPTION: SEQ ID CCGGTCGTCC TGG 13 INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: CAATTCCGGT GTACTCACCG GTTCC 263 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 9 base pairs TYPE: nucleic acid STRANDEDNESS: sinale TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a spacer bearing a Cy3 amidite label." (ix) FEATURE: NAME/KEY: modified base LOCATION: 2..3 OTHER INFORMATION: /mod base= OTHER /note= "The residues at these positions have an amino group added." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: NTTCCAGAG 9 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyguanosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(2, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(3..4, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: o* NAME/KEY: miscdifference LOCATION: replace(5, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(6, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5 '-O-(l-Thiomonophosphate)." 264 (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(7..8, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(9, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(10, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: GTAATCTTAC CAACGCTAAC GAGCGTCTTG INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(1. OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(3, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(4..5, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(6..8, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxythymidine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(9, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyguanosine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: miscdifference LOCATION: replace(10, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." -265- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: CCTAATTTGC CAGTTACAAA ATAAACAGCC INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: GGGAAAGTCC TCGGAGCCGC GCGGGACGAG CGTGGGGGCC CG INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 963 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..960 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: o r 2 GCT AGC ATG Ala Ser Met GGT GGA CAG CAA Gly Gly Gln Gln ATG GGT CGG ATC AAT TCG GGG Met Gly Arg Ile Asn Ser Gly 10 ATG CTG CCC CTC TTT GAG CCC AAG Met Leu Pro Leu Phe Glu Pro Lys CGG GTC CTC CTG Arg Val Leu Leu GTG GAC GGC Val Asp Gly CTC ACC ACC Leu Thr Thr CAC CAC CTG His His Leu GCC TAC CGC ACC TTC CAC GCC CTG AAG Ala Tyr Arg Thr Phe His Ala Leu Lys AGC CGG Ser Arg GGG GAG CCG GTG Gly Glu Pro Val GCG GTC TAC GGC Ala Val Tyr Gly GCC AAG AGC CTC Ala Lys Ser Leu r AAG GCC CTC AAG Lys Ala Leu Lys GAG GAC GGG GAC GCG GTG ATC GTG GTC TTT GAC Glu Asp Gly Asp Ala Val Ile Val Val Phe Asp 75 GCC AAG GCC CCC Ala Lys Ala Pro TTC CGC CAC GAG Phe Arg His Glu GCC TAC GGG GGG TAC AAG GCG Ala Tyr Gly Gly Tyr Lys Ala 90 GGC CGG GCC CCC ACG CTC GTC CCG CGC GGC TCC GAG GAC TTT CCC CGG Gly Arg Ala Pro Thr Leu Val Pro Arg Gly Ser Glu Asp Phe Pro Arg 100 105 110 CAA CTC GCC Gln Leu Ala 115 CTC ATC AAG GAG Leu Ile Lys Glu GTG GAC CTC CTG Val Asp Leu Leu CTG GCG CGC Leu Ala Arg CTC GAG Leu Glu 130 GTC CCG GGC TAC Val Pro Gly Tyr GCG GAC GAC GTC Ala Asp Asp Val GCC AGC CTG GCC Ala Ser Leu Ala 266-

AAG

Lys 145

AAA

Lys

GAG

Glu

AGG

Arg

GAC

AST)

CTT

Leu 225

CGG

Arg

CTG

Leu

GAG

Glu

GCC

Ala

CTT

Leu 305

TGA

AAG GCG GAA Lys Ala Glu AAG GAG GGC Lys Glu Gly 150 TAC GAG GTC Tyr Glu Val CGC ATC CTC ACC CC Arg Ile Leu Thr Ala 155 CAG CTC Gin LP., 165 ATC ACC Ile Thr TGG GCC Trp Ala GGG GTC Gly Val TGG GGG Trp Gly 230 GCC ATC Ala Ile 245 TGG GAC Trp Asp GCC AAA Ala Lys AGG CTT Arg Leu CCC AAG Pro Lys 310

ATC

TIC

TGG

Trp

CTG

Leu

GAG

Glu

CTC

Leu 235

CTG

Leu

CC

Arg

GAC

Asp

CTC

Leu

CAC

His 315

CTC

TAC

190

GAC

Asp

GCG

Al a

AAC

Asn

ATG

Met

CTG

Leu 270

AGG

Arg

GAG

Giu

CAC

His

GAC

Asp 160

CCC

P I.u

CTG

Leu

TCC

Ser

AAG

Lys

GAC

Asp 240

GAT

Asp

CTG

Leu

AGG

Arg

GGC

Gly

CAC

His 320 480 528 576 624 672 720 768 816 864 912 960 963

CS...

INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 320 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Met Ala Ser Met Thr Gly Gly Gln Gin Met Giy Arg Ile Asn Ser Gly 1 5 10 Met Leu Pro Leu Phe Giu Pro Lys Gly Arg Val Leu Leu Val Asp Gly 25 His His Leu Aia Tyr Arg Thr Phe His Ala Leu Lys Gly Leu Thr Thr 40 Ser Arg Gly Glu Pro Vai Gin Ala Val Tyr Gly Phe Ala Lys Ser Leu 55 267 Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val Val Phe Asp 70 75 Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly Tyr Lys Ala 90 Gly Ary Ala Pro Thr Leu Val Pro Arg Gly Ser Glu Asp Phe Pro Arg 100 105 110 Gin Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg 115 120 125 Leu Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala 130 135 140 Lys Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp 145 150 155 160 Lys Asp Leu Tyr Gin Leu Leu Ser Asp Arg Ile His Val Leu His Pro 165 170 175 Glu Gly Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu 180 185 190 Arg Pro Asp Gin Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser 195 200 205 Asp Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys 210 215 220 Leu Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp 225 230 235 240 Arg Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp S* 245 250 255 Leu Lys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu 260 265 270 Glu Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg 275 280 285 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 290 295 300 Leu Leu Glu Ser Pro Lys Ala Ala Leu Glu His His His His His His 305 310 315 320 INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs S(B) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: CGATCTCCTC GGCCACCTCC INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -268 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: CCAGCTCGTT GTGGACCTGA INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 2505 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2499 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: r r r r rr ATG AAT TCG GGG Met Asn Ser Gly 1 CTG CCC CTC TTT GAG CCC AAG GGC CGG Leu Pro Leu Phe Glu Pro Lys Gly Arg 10 GTC CTC Val Leu r r r CTG GTG GAC Leu Val Asp GGC CTC ACC Gly Leu Thr CAC CAC CTG GCC His His Leu Ala CGC ACC TTC CAC Arg Thr Phe His GCC CTG AAG Ala Leu Lys TAC GGC TTC Tyr Gly Phe ACC AGC CGG GGG Thr Ser Arg Gly CCG GTG CAG GCG Pro Val Gln Ala GCC AAG Ala Lys AGC CTC CTC AAG Ser Leu Leu Lys CTC AAG GAG GAC Leu Lys Glu Asp GAC GCG GTG ATC Asp Ala Val Ile GTC TTT GAC GCC Val Phe Asp Ala GCC CCC TCC TTC Ala Pro Ser Phe CAC GAG GCC TAC His Glu Ala Tyr GGG TAC AAG GCG Gly Tyr Lys Ala GGC CGG GCC CCC ACG CCG GAG GAC TTT CCC CGG CAA Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gin 90 CTC GCC CTC Leu Ala Leu AAG GAG CTG GTG Lys Glu Leu Val CTC CTG GGG CTG Leu Leu Gly Leu GCG CGC CTC Ala Arg Leu 110 GAG GTC CCG GGC TAC GAG GCG GAC GAC GTC CTG GCC AGC CTG GCC AAG Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys 115 120 125 269 AAG GCG Lys Ala 130 GAA AAG GAG GGC TAC GAG GTC CGC ATC Glu Lys Giu Gly Tyr Glu Val Arg Ile 135 ACC GCC GAC AAA Thr Ala Asp Lys CT? TAC CAG CTC TCC GAC CGC ATC Sc s F l GTC CTC CAC CCC Val Leu His Pro GGG TAC CTC ATC Gly Tyr Leu Ile CCG GCC TGG CTT Pro Ala Trp Leu GAA AAG TAC GGC Glu Lys Tyr Gly CTG AGG Leu Arg 175 CCC GAC CAG Pro Asp Gin AAC CTT CCC Asrn Leu Pro 195 GCC GAC TAC CGG Ala Asp Tyr Arg CTG ACC GGG GAC Leu Thr Gly Asp GAG TCC GAC Glu Ser Asp 190 576 GGG GTC AAG GGC Gly Val Lys Gly ATC GGG GAG AAG ACG GCG AGG AAG CT? Ile Gly Glu Lys Thr Ala Arg Lys Leu 200 205 CTG GAG Leu Glu 210 GAG TGG GGG AGC Glu Trp Gly Ser GAA GCC CTC CTC Glu Ala Leu Leu AAC CTG GAC CGG Asn Leu Asp Arg AAG CCC GCC ATC Lys Pro Ala Ile GAG AAG ATC CTG Glu Lys Ile Leu CAC ATG GAC GAT His Met Asp Asp AAG CTC TCC TGG Lys Leu Ser Trp CTG GCC AAG GTG Leu Ala Lys Val ACC GAC CTG CCC Thr Asp Leu Pro CTG GAG Leu Glu 255 GTG GAC TTC Val Asp Phe ?TT CTG GAG Phe Leu Glu 275 AAA AGG CGG GAG Lys Arg Arg Glu GAC CGG GAG AGG Asp Arg Glu Arg CTT AGG GCC Leu Arg Ala 270 TC GGC CTT Phe Gly Leu 816 AGG CTT GAG TTT Arg Leu Glu Phe AGC CTC CTC CAC Ser Leu Leu His CTG GAA Leu Glu 290 AGC CCC AAG GCC Ser Pro Lys Ala GAG GAG GCC CCC Glu Glu Ala Pro CCC CCG CCG GAA Pro Pro Pro Glu GCC TTC GTG GGC Ala Phe Val Gly GTG CTT TCC CGC Val Leu Ser Arg GAG CCC ATG TGG Glu Pro Met Trp GAT CT? CTG GCC Asp Leu Leu Ala GCC GCC GCC AGG Ala Ala Ala Arg GGC CGG GTC CAC Gly Arg Val His CGG GCC Arg Ala 335 CCC GAG CCT Pro Giu Pro CTC GCC AAA Leu Ala Lys AAA GCC CTC AGG Lys Ala Leu Arg CTG AAG GAG GCG Leu Lys Glu Ala CGG GGG CT? Arg Gly Leu 350 CT? CCC CTC Leu Gly Leu GAC CTG AGC CTT Asp Leu Ser Val GCC CTG AGC GAA Ala Leu Arg Glu 1008 1056 1104 1152 1200 CCG CCC Pro Pro 370 CCC GAC GAC CCC Gly Asp Asp Pro CTC CTC CCC TAC Leu Leu Ala Tyr CTG GAC CCT TCC Leu Asp Pro Ser ACC ACC CCC GAG Thr Thr Pro Glu GTG GCC CGG CGC Val Ala Arg Arg GGC GCC GAG TCC Gly Gly Glu Trp, 270 GAG GAG GCG GGG Glu Glu Ala Gly CTG TGG GGG AGG T,Pii r1A r 420 GAG GTG GAG AGG Glu Val Glu Arg 435 CGG GCC GCC CTT Arg Ala Ala Leu GAG AGG CTC TTC Giu Arg Leu Phe GCC AAC Ala Asn 415 =T GAG GGG GAG AGG CTC CTT TGG Aiyj Leu Leu Trp CTT TAC CGG Leu Tyr Arg 430 CCC CTT TCC Pro Leu Ser GTC CTG GCC CAC ATG GAG GCC ACG Val Leu Ala His Met Glu Ala Thr 445 GGG GTG Gly Val 450 CGC CTG GAC GTG Arg Leu Asp Val GCC TAT CTC AGG GCC TTG TCC CTG GAG GTG Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val 455 460 GAG GAG ATC GCC Glu Glu Ile Ala CTC GAG GCC GAG Leu Giu Ala Glu GTC TTC CGC CTG GCC GGC Val Phe Arg Leu Ala Gly 475 480 CAC CCC TTC AAC Hius Pro Phe Asn GAC GAG CTA GGG Asp Glu Leu Gly 500 AAC TCC CGG GAC Asn Ser Arg Asp CTG GAA AGG GTC Leu Glu Arg Val CTC =T Leu Phe 495 CTT CCC GCC ATC Leu Pro Ala Ile GGC AAG ACG GAG AAG ACC GGC AAG Gly Lys Thr Glu Lys Thr Gly Lys 505 510 CGC TCC ACC Arg Ser Thr 515 AGC GCC GCC GTC Ser Ala Ala Val GAG GCC CTC CGC Glu Ala Leu Arg GCC CAC CCC Ala His Pro ATC GTG Ile Val 530 GAG AAG ATC CTG Glu Lys Ile Leu CAG TAC CGG GAG CTC ACC AAG CTG AAG AGC Gin Tyr Arg Glu Leu Thr Lys Leu Lys Ser 535 540 1246 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 TAC ATT GAC CCC Tyr Ile Asp Pro CCG GAC CTC ATC Pro Asp Leu Ile CCC AGG ACG GGC Pro Arg Thr Gly CTC CAC ACC CGC Leu His Thr Arg AAC CAG ACG GCC Asn Gin Thr Ala GCC ACG GGC AGG Ala Thr Gly Arg CTA AGT Leu Ser 575 AGC TCC GAT Ser Ser Asp CAG AGG ATC Gin Arg Ile 595 A-AC CTC CAG AAC Asn Leu Gln Asn CCC GTC CGC ACC Pro Val Arg Thr CCG CTT GGG Pro Leu Gly 590 CTA TTG GTG Leu Leu Val CGC CGG GCC TTC Arg Arg Ala Phe GCC GAG GAG GGG Ala Giu Giu Gly GCC CTG GAC TAT AGC CAG ATA GAG CTC AGG GTG CTG GCC CAC CTC TCC Ala Leu Asp Tyr Ser Gin Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620 GAC GAG AAC CTG Asp Glu Asn Leu CGG GTC TTC CAG Arg Val Phe Gin GGG CGG GAC ATC Gly Arg Asp Ile ACG GAG ACC GCC AGC TGG ATG TTC GGC Thr Giu Thr Ala Ser Trp Met Phe Gly 645 CCC CGG GAG GCC Pro Arg Glu Ala GTG GAC Val Asp 655 CCC CTG ATG Pro Leu Met CGG GCG GCC AAG Arg Ala Ala Lys ATC AAC TTC GGG Ile Asn Phe Gly GTC CTC TAC Val Leu Tyr 670 271 GGC ATG TCG GCC CAC CGC CTC TCC CAG GAG CTA Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu 675 680 GAG GCC CAG GCC TTC ATT GAG CGC TAC TTT CAG Glu Ala Gin Ala Phe Ile Glu Arq Tyr Phe Gin b~u 695 CGG GCC TGG ATT GAG AAG ACC CTG GAG GAG GGC Arg Ala Trp Ilie Glu Lys Thr Leu Giu Glu Gly 705 710 715 GTG GAG ACC CTC TTC GGC CGC CGC CGC TAC GTG Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val 725 730 COG GTG AAG AGC GTG CGG GAG GCG GCC GAG CGC Arg Val Lys Ser Val Arg Giu Aia Ala Giu Arg 740 745 CCC GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG Pro Val Gin Gly Thr Ala Ala Asp Leu Met Lys 755 760 CTC TTC CCC AGO CTG GAG GAA ATG 000 GCC AGO Leu Phe Pro Arg Leu 0Th Olu Met Gly Ala Arg 770 775 CAC AAC GAG CTG GTC CTC GAG GCC CCA AAA GAG His Asn Olu Leu Val Leu Oiu Ala Pro Lys Glu 785 790 795 GCC CGG CTG 0CC AAG GAG GTC ATG GAG GOG GTG Ala Arg Leu Ala Lys Oiu Val Met Giu Gly Val 805 810 CCC CTG GAG GTG GAG GTG GOG ATA GGG GAG GAC Pro Leu Glu Val Olu Val Gly Ile Oly Glu Asp 820 825 GAG TGATAO Glu INFORMATION FOR SEQ ID NO:107: GCC ATC CCT TAC GAG Ala Ile Pro Tyr Giu 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2505 Met Leu Gly Ala Val1 Asn Val1 Leu Lys Val SEQUENCE CHARACTERISTICS: LENGTH: 833 amino acids TYPE: amino acid TOPOLOGY: linear i) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID Ser Gly Met Leu Pro Leu Phe Glu 10 Asp Gly His His Leu Ala Tyr Arg 25 Thr Thr Ser Arg Gly Glu Pro Val 40 Ser Leu Leu Lys Ala Leu Lys Giu 55 Phe Asp Ala Lys Ala Pro Ser Phe 70 NO: 107: Pro Lys Gly Arg Val Leu Thr Phe His Ala Leu Lys Gln Ala Val Tyr Gly Phe Asp Gly Asp Ala Vai Ile Arg His Glu Ala Tyr Giy 75 272 Giy Tyr Lys Ala Giy Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gin 90 Leu Uliu Lys Asp 145 Gly Pro Asn Leu Leu 225 Lys Val1 Phe Leu Gly 305 Asp Pro Leu Pro Asn 385 Glu Leu Giu Leu Ile 100 Pro Giy 115 Giu Lys Tyr Gin Leu Ile Gin Trp 180 Pro Gly 195 Giu Trp Pro Ala Ser Trp Phe Aia 260 Giu Arg 275 Ser Pro Phe Vai Leu Aia Pro Tyr 340 Lys Asp 355 Giy Asp Thr Pro Aia Giy Gly Arg 420 Giu Arg 435 Glu Glu Giy Leu 150 Pro Asp Lys Ser Arg 230 Leu Arg Giu Ala Phe 310 Ala Ala Ser Pro Giy 390 Arg Glu Leu Leu Aila Tyr 135 Ser Aia Tyr Gly Leu 215 Giu Al a Arg Phe Leu 295 Val Al a Leu Val Met 3.75 Val1 Ala Gly Ser Val Asp 120 Glu Asp Tr-p Arg Ile 200 Glu Lys Lys Glu Gi y 280 Glu Leu Al a Arg Leu 360 Leu Ala Al a Glu Ala 440 Leu Gly Leu Ala Ile Leu 140 H-is Val Glu Lys Thr Gly Lys Thr Leu Lys 220 Ala His 235 Thr Asp Arg Glu Leu His Pro Trp, 300 Lys Glu 315 Gly Arg Lys Glu Arg Glu Tyr Leu 380 Tyr Gly 395 Glu Arg Leu Leu Ala His Leu Ser 125 Thr Leu Tyr Asp Ala 205 Asn Met Leu Arg Glu 285 Pro Pro Val1 Ala Gly 365 Leu Gly Leu Trp, Met 445 Ala 110 Leu Al a His Gly Glu 190 Arg Leu Asp Pro Leu 270 Phe Pro Met His Arg 350 Leu Asp Glu Phe Leu 430 Glu Arg Ala Asp Pro Leu 175 Ser Lys Asp Asp Le u 255 Arg Gly Pro Trp, Arg 335 Gi y Gly Pro Trp Ala 415 Tyr Ala Leu Lys Lys Glu 160 Arg Asp Leu Arg Leu 240 Glu Al a Leu Gi u Ala 320 Al a Leu Leu Ser Thr 400 Asn Arg Thr 273 Gly Val Arg Leo Asp Val Ala Tyr Leu Arg Ala 450 455 Leo Ser Leu Glu Val 460 Ala Glu Glu Ile Ala Arg 465 His Asp Arg Ile Thr 545 Leu Ser Gln Al a Gi y 625 Thr Pro Gly Glu Arg 705 Val Arg Pro Leu His 785 Asn Gly 500 Ser Lys Asp Arg Pro 580 Arg Tyr Asn Al a Arg 660 Ala Al a Ile Leu Ser 740 Gly Arg Leo Leu 485 Leo Al a Ile Pro Phe 565 Asr.

Arg Ser Leu Ser 645 Arg His Phe Glu Phe 725 Val Thr Leo Val1 Lys 805 470 Asn Pro Al a Leo Leo 550 Asn Leo Ala Gin Ile 630 Trp Ala Arg Ile Lys 710 Gly Arg Ala Glu Leo 790 Leo Glu Ser Arg Ala Ile Val Leo 520 Gin Tyr 535 Pro Asp Gin Thr Gin Asn Phe Ile 600 Ile Glu 615 Arg Val Met Phe Ala Lys Leo Ser 680 Glu Arg 695 Thr Leo Arg Arg Glu Ala Ala Asp 760 Gbu Met 775 Giu Ala Ala Asp Gly 505 Glu Arg Leo Ala Ile 585 Al a Leo Phe Gly Thr 665 Gln Tyr Glu Arg Ala 745 Leu Gly Pro Glu Gin 490 Lys Ala Glu Ile Thr 570 Pro Glu Arg Gin Val 650 Ile Glu Phe Glu Tyr 730 Glu Met Ala Lys di y 810 Vai 475 Leo Thr Leo Leo His 555 Al a Val Glu Val diu 635 Pro Asn Leo din Gly 715 Val Arg Lys Arg Glu 795 Val Phe Arg Gbu Arg Glu Lys Arg Gbu 525 Thr Lys 540 Pro Arg Thr Giy Arg Thr Gly Trp 605 Leo Ala 620 Gly Arg Arg dlu Phe Gly Ala Ile 685 Ser Phe 700 Arg Arg Pro Asp Met Ala Leo Ala 765 Met Leo 780 Arg Ala Tyr Pro Ala Gly 480 Leo Phe 495 Gly Lys His Pro Lys Ser Gly Arg 560 Leo Ser 575 Leu Gly Leo Val Leo Ser Ile His 640 Val Asp 655 Leo Tyr Tyr Glu Lys Val Gly Tyr 720 Gbu Ala 735 Asn Met Val Lys Gin Val Ala Val 800 Ala Val 815 Ala Arg Leu Ala Glu Val Met dlu 274 Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 .o.

Glu INFORMATION FOR SEQ TO SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: GGGATACCAT GGGAGTGCAG TTTGG INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: GGTAAATTTT TCTCGTCGAC ATCCCAC INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 981 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..978 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: ATG GGA GTG CAG TTT GGT GAT TTT ATT CCA AAA AAT ATT Met Gly Val Gln Phe Gly Asp Phe Ile Pro Lys Asn Ile 1 5 10 GAA GAT TTA AAA GGG AAA AAA GTA GCT ATT GAT GGA ATG Glu Asp Leu Lys Gly Lys Lys Val Ala Ile Asp Gly Met 20 25 TAT CAG TTT TTA ACA TCT ATA CGT TTG AGA GAT GGT TCT Tyr Gin Phe Leu Thr Ser Ile Arg Leu Arg Asp Gly Ser 40 AAT AGA AAA GGA GAG ATA ACC TCA GCA TAT AAC GGA GTT Asn Arg Lys Gly Glu Ile Thr Ser Ala Tyr Asn Gly Val 55 ACC ATA CAT TTG TTA GAG AAT GAT ATA ACT CCA ATC TGG Thr Ile His Leu Leu Glu Asn Asp Ile Thr Pro Ile Trp 70 ATC TCC TTT Ile Ser Phe AAT GCA TTA Asn Ala Leu CCA TTG AGA Pro Leu Arg TTT TAT AAA Phe Tyr Lys GTT TTT Val Phe 275 GGT GAG CCA CCA AAG TTA AAG GAG AAA ACA AGG AAA GTT AGG AGA GAG Gly Glu Pro Pro Lys Leu Lys Glu Lys Thr Arg Lys Val Arg Arg Glu 90 ATG AAA GAG Met Lys Glu GAT TTT GAA Asp Phe Glu 115 GCT GAA CTT AAG Ala Glu Leu Lys AAA GAG GCA ATT Lvs Glu Ala TIe AAA AAG GAG T -1 110 TAT CTA ACT Tyr Leu Thr GAA GCT GCT AAG Glu Ala Ala Lys GCA AAG AGG GTT Ala Lys Arg Val CCG AAA Pro Lys 130 ATG GTT GAA AAC Met Val Glu Asn TGC AAA TAT TTG TTA AGT TTG ATG GGC ATT Cys Lys Tyr Leu Leu Ser Leu Met Gly Ile 135 140 CCG TAT GTT GAA GCT CCC TCT GAG GGA GAG GCA CAA GCA AGC TAT ATG Pro Tyr Val Glu Ala Pro Ser Glu Gly Glu Ala Gin Ala Ser Tyr Met 145 150 155 160 GCA AAG AAG GGA Ala Lys Lys Gly GTT TGG GCA GTT Val Trp Ala Val AGT CAA GAT TAT Ser Gin Asp Tyr GAT GCC Asp Ala 175 TTG TTA TAT Leu Leu Tyr GAG ATG CCA Glu Met Pro 195 GCT CCG AGA GTT Ala Pro Arg Val AGA AAT TTA ACA Arg Asn Leu Thr ACT ACA AAG Thr Thr Lys 190 GAT TTA AGA Asp Leu Arg GAA CTT ATT GAA Glu Leu Ile Glu AAT GAG GTT TTA Asn Glu Val Leu ATT TCT Ile Ser 210 TTG GAT GAT TTG Leu Asp Asp Leu GAT ATA GCC ATA Asp Ile Ala Ile ATG GGA ACT GAC Met Gly Thr Asp AAT CCA GGA GGA Asn Pro Gly Gly GTT AAA GGA ATA GGA TTT AAA AGG GCT TAT GAA Val Lys Gly Ile Gly Phe Lys Arg Ala Tyr Glu 230 235 240 720 TTG GTT AGA AGT Leu Val Arg Ser GTA GCT AAG GAT Val Ala Lys Asp TTG AAA AAA GAG Leu Lys Lys Glu GTT GAA Val Glu 255 TAC TAC GAT Tyr Tyr Asp AAC TAT TCA Asn Tyr Ser 275 ATT AAG AGG ATA Ile Lys Arg Ile AAA GAG CCA AAG Lys Glu Pro Lys GTT ACC GAT Val Thr Asp 270 ATT ATA AAA Ile Ile Lys TTA AGC CTA AAA Leu Ser Leu Lys CCA GAT AAA GAG Pro Asp Lys Glu TTC TTA Phe Leu 290 GTT GAT GAA AAT Val Asp Glu Asn TTT AAT TAT GAT Phe Asn Tyr Asp

AGG

Arg 300 GTT AAA AAG CAT Val Lys Lys His GTT GAT AAA CTC TAT AAC TTA ATT GCA AAC AAA ACT AAG CAA AAA ACA Val Asp Lys Leu Tyr Asn Leu Ile Ala Asn Lys Thr Lys Gin Lys Thr 305 310 315 320 TTA GAT GCA TGG Leu Asp Ala Trp TTT AAA TAA Phe Lys 325 INFORMATION FOR SEQ ID NO:111: SEQUENCE CHARACTERISTICS: LENGTH: 326 amino acids TYPE: amino acid TOPOLOGY: linear -276 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:111: 4* S. *5 Met 1 Gi u Tyr Asn Thr Gly Met Asp Pro Pro 145 Ala Leu Glu Ile Tyr 225 Leu Tyr Asn Phe Val1 305 Leu Gly Asp Phe Ile Pro Lys Asn Ile Ile Ser Phe 277 INFORMATION FOR SEQ ID NO:112: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid ^mA^^DEDNES S.iiTjIe TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: GAGGTGATAC CATGGGTGTC C INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: GAAACTCTGC AGCGCGTCAG INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 1023 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1020 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: ATG GGT GTC CCA ATT GGT GAG ATT ATA CCA AGA AAA r Met Gly Val Pro Ile Gly Glu Ile Ile *.aa a a a.

Pro Arg Lys 10 ATC GAC GCT Ile Asp Ala GAA ATT GAG TTA Glu Ile Glu Leu CTT AAT GCA ATC Leu Asn Ala Ile GAA AAC CTA Glu Asn Leu TAC CAA TTT Tyr Gln Phe GAT TCA AAG Asp Ser Lys GGG AAA AAA ATC Gly Lys Lys Ile CCA CTT ATG Pro Leu Met TCC ACA ATA Ser Thr Ile CAG AAA GAT GGA Gln Lys Asp Gly CAC CTA AGC GGG His Leu Ser Gly 144 GGT AGA ATA Gly Arg Ile CTC TTT TAC AGG Leu Phe Tyr Arg ACA ATA Thr Ile AAC CTA ATG Asn Leu Met GAG GCT GGA ATA AAA CCT GTG TAT GTT Glu Ala Gly Ile Lys Pro Val Tyr Val 75 TTC AAA AAG AAA GAG CTC GAA AAA AGA Phe Lys Lys Lys Glu Leu Glu Lys Arg TTT GAT Phe Asp AGA GAA Arg Glu GGA GAA CCT CCA Gly Glu Pro Pro 278 GCG AGA GAG Ala Arg Giu GAG ATA GAG Giu Ile Giu 115 GCT GAA GAA AAG Ala Giu Giu Lys AGA GAA GCA CTT Arg Giu Ala Leu GAA AAA GGA Glu Lys Gly 110 AGG GTA AAT Arg Val Asn GAA GCA AGA AAA Giu Ala Arg Lys GCC CAA AGA GCA Ala Gin Arg Ala GAA ATG Giu Met 130 CTC ATC GAG GAT GCA AAA AAA CTC TTA Leu Ile Giu Asp Ala Lys Lys Leu Leu 135 CTT ATG GGA ATT Leu Met Giy Ile 432 CCT ATA GTT CAA GCA Pro Ile Val Gin Ala 145 AGC GAG GGA GAG Ser Giu Gly Giu CAA GCT GCA TAT Gin Ala Ala Tyr GCC GCA AAG GGG Ala Ala Lys Giy GTG TAT GCA TCG Val Tyr Ala Ser AGT CAA GAT TAC Ser Gin Asp Tyr GAT TCC Asp Ser 175 CTA CTZ' TTT Leu Leu Phe GCT CCA AGA CTT Ala Pro Arg Leu AGA AAC TTA ACA Arg Asn Leu Thr ATA ACA GGA Ile Thr Gly 190 57 6 AAA AGA AAG TTG CCT GGG AAA AAT GTC TAC GTC GAG ATA AAG CCC GAG Lys Arg Lys Leu Pro Giy Lys Asn Val Tyr Val Giu Ile Lys Pro Giu 195 200 205 TTG ATA Leu Ile 210 ATT TTG GAG GAA Ile Leu Giu Giu CTC AAG GAA TTA Leu Lys Giu Leu CTA ACA AGA GAA Leu Thr Arg Giu CTC ATT GAA CTA Leu Ile Giu Leu ATC CTC GTT GGA Ile Leu Val Gly GAC TAC AAC CCA Asp Tyr Asn Pro 720 768 0 0 GGA ATA AAG GGC ATA GGC CTT AAA AAA GCT TTA GAG ATT GTT AGA CAC Gly Ile Lys Gly Ilie Gly Leu Lys Lys Ala Leu Giu Ile Val Arg His 245 250 255 TCA AAA GAT Ser Lys Asp CTA GCA AAG TTC Leu Ala Lys Phe

CAA

Gin 265 AAG CAA AGC GAT Lys Gin Ser Asp GTG GAT TTA Val Asp Leu 270 *5S TAT GCA ATA AAA GAG TTC TTC CTA AAC CCA CCA GTC ACA GAT AAC TAC Tyr Ala Ilie Lys Glu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr 275 280 285 AAT TTA Asn Leu 290 GTG TGG AGA GAT Val Trp Arg Asp GAC GAA GAG GGA Asp Giu Giu Gly CTA AAG TTC TTA Leu Lys Phe Leu GAC GAG CAT GAC Asp Glu His Asp TTT ACT GAG GAA AGA GTA AAG AAT GGA TTA GAG Phe Ser Giu Glu Arg Val Lys Asn Gly Leu Glu 310 315 320 AGG CTT AAG AAG Arg Leu Lys Lys ATC AAA ACT GGA Ile Lys Ser Gly

AAA

Lys 330 CAA TCA ACC CTT Gin Ser Thr Leu GAA ACT Giu Ser 335 1008 1023 TGG TTC AAG Trp Phe Lys AGA TAA Arg 340 279 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOfl.r~'CULE TYPE: pzar (xi) SEQUENCE DESCRIPTION: SEQ ID N0:115: Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Glu Ile Giu Leu 1 5 10 Giu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile 25 Tyr Gin Phe Leu Ser Thr Ile Arg Gin Lys Asp Gly Thr Pro Leu Met 40 Asp Ser Lys Giy Arg Ile Thr Ser His Leu Ser Giy Leu Phe Tyr Arg 55 Thr Ile Asn Leu Met Giu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp 70 75 Gly Giu Pro Pro Glu Phe Lys Lys Lys Glu Leu Giu Lys Arg Arg Giu 90 Ala Arg Glu Glu Ala Giu Glu Lys Trp Arg Giu Ala Leu Glu Lys Gly 100 105 110 esGlu Ile Glu Glu Ala Arg Lys Tyr Ala Gin Arg Ala Thr Arg Val Asri 0115 120 125 Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Giu Leu Met Gly Ile 130 135 140 *aSPro Ile Val Gin Ala Pro Ser Glu Gly Giu Ala Gin Ala Ala Tyr Met "00:69* 145 150 155 160 Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gin Asp Tyr Asp Ser 165 170 175 gof* .0 Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly 180 185 190 Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu 195 200 205 Leu Ile Ile Leu Glu Giu Val Leu Lys Glu Leu Lys Leu Thr Arg Giu 210 215 220 Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly 225 230 235 240 .Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Giu Ile Val Arg His *245 250 255 Ser Lys Asp Pro Leu Ala Lys Phe Gin Lys Gin Ser Asp Val Asp Leu 260 265 270 Tyr Ala Ile Lys Giu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr 275 280 285 Asn Leu Val Trp Arg Asp Pro Asp Giu Giu Gly Ile Leu Lys Phe Leu 290 295 300 Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly Leu Giu 305 310 315 320.

280 Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gin Ser Thr Leu Glu Ser 325 330 335 Trp Phe Lys Arg 340 INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: GATACCATGG GTGTCCCAAT TGGTG INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: TCGACGTCGA CTTATCTCTT GAACCAACTT TCAAGGG 37 INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: AGCGAGGGAG AGGCCCAAGC INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: GCCTATGCCC TTTATTCCTC C 21 281 INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: TGGTCGCTGT CTCGCTGAAA GCGAGACAGC GTG 33 INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: TGCTCTCTGG TCGCTGTCTG AAAGACAGCG INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: CGAGAGACCA CGCT 14 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: TTTTCCAGAG CCTAATGAAA TTAGGCTCTG GAAAGACGCT CGTG 44 INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" **282 S- 282 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: AACGAGCGTC TTTG 14 INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: AACGAGCGTC ATTG 14 INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: TTTTTTTTTA ATTAGGCTCT GGAAAGACGC TCGTGAAACG AGCGTCTTTG INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: TTTTCCAGAG CCTAATG 17 INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: TGGCTATAGR CCAGGGCCAC -283 INFORMATION FOR SEQ ID NO:129: SEQUENCE CHARACTERISTICS: LENGTH: 2505 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2499 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: ATG AAT TCG GGG ATG CTG CCC CTC TTT Met Asn Ser Gly Met Leu Pro Leu Phe 1 CTG GTG GAC GGC CAC CAC CTG GCC TAC Leu Val Asp Gly His His Leu Ala Tyr GGC CTC ACC ACC AGC CGG GGG GAG CCG Gly Leu Thr Thr Ser Arg Gly Glu Pro GCC AAG AGC CTC CTC AAG GCC CTC AAG Ala Lys Ser Leu Leu Lys Ala Leu Lys GTG GTC TTT GAC GCC AAG GCC CCC TCC Val Val Phe Asp Ala Lys Ala Pro Ser GGG TAC AAG GCG GGC CGG GCC CCC ACG Gly Tyr Lys Ala Gly Arg Ala Pro Thr CTC GCC CTC ATC AAG GAG CTG GTG GAC Leu Ala Leu Ile Lys Glu Leu Val Asp 100 105 GAG GTC CCG GGC TAC GAG GCG GAC GAC Glu Val Pro Gly Tyr Glu Ala Asp Asp 115 120 AAG GCG GAA AAG GAG GGC TAC GAG GTC Lys Ala Glu Lys Glu Gly Tyr Glu Val 130 135 GAC CTT TAC CAG CTC CTT TCC GAC CGC Asp Leu Tyr Gln Leu Leu Ser Asp Arg 145 150 GGG TAC CTC ATC ACC CCG GCC TGG CTT Gly Tyr Leu Ile Thr Pro Ala Trp Leu 165 CCC GAC CAG TGG GCC GAC TAC CGG GCC Pro Asp Gln Trp Ala Asp Tyr Arg Ala I rf 1 or CCC AAG GGC CGG GTC CTC Pro Lys Gly Arg Val Leu 528 AAC CTT CCC Asn Leu Pro 195 GGG GTC AAG GGC ATC GGG Gly Val Lys Gly Ile Gly 624 -284- CTG GAG Leu Glu 210 GAG TGG GGG AGC Giu TI-p Gly Ser GAA GCC CTC CTC Glu Ala Leu Leu AAC CTG GAC CGG Asn Leu Asp Arg CTG AAG CCC GCC ATC CGG GAG AAG ATC CTG GCC CAC ATG GAC GAT CTG Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu 225 230 235 240 AAG CTC TCC TGG Lys Leu Ser Trp CTG GCC AAG GTG Leu Ala Lys Val ACC GAC CTG CCC Thr Asp Leu Pro CTG GAG Leu Glu 255 GTG GAC TTC Val Asp Phe AAA AGG CGG GAG Lys Arg Arg Glu CCC GAC CGG GAG AGG CTT AGG GCC Pro Asp Arg Glu Arg Leu Arg Ala 265 270 TTT CTG GAG AGG Phe Leu Glu Arg 275 CTT GAG T Leu Glu Phe AGC CTC CTC CAC Ser Leu Leu His TTC GGC CTT Phe Gly Leu CTG GAA Leu Glu 290 AGC CCC AAG GCC Ser Pro Lys Ala GAG GAG GCC CCC Glu Glu Ala Pro CCC CCG CCG GAA Pro Pro Pro Glu

GGG

Gly 305 GCC TTC GTC GGC Ala Phe Val Gly CTC CTT TCC CGC Val Leu Ser Arg GAG CCC ATG TGG Glu Pro Met Trp GAT CTT CTG GCC Asp Leu Leu Ala GCC GCC GCC AGG Ala Ala Ala Arg GGC CGG GTC CAC Gly Arg Val His CGG GCC Arg Ala 33S CCC GAG CCT Pro Glu Pro CTC GCC AAA Leu Ala Lys 355 TAT AAA GCC CTC AGG CAC CTG AAG GAG GC CGG GGG CTT Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu 340 345 350 GAC CTG AGC GTT Asp Leu Ser Val GCC CTG AGG GAA Ala Leu Arg Glu CTT GGC CTC Leu Gly Leu CCG CCC GGC GAC GAC CCC ATG CTC CTC GCC TAC CTC CTG GAC CCT TCC Pro Pro Cly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380 ACC ACC CCC GAG Thr Thr Pro Glu GTG GCC CGG CGC Val Ala Arg Arg TAC GGC GGG GAG TGG ACG Tyr Gly Gly Glu Trp Thr 395 400 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440

C

GAG GAG GCG GGG GAG CGG GCC GCC CTT Glu Glu Ala Gly Glu Arg Ala Ala Leu GAG AGG CTC TTC Glu Arg Leu Phe GCC AAC Ala Asn 415 CTG TGG GGG Leu Trp Gly GAG GTG GAG Glu Val Glu 435 CTT GAG GGG GAG Leu Glu Gly Glu GAG AGG CTC =T TGG CTT TAC CGC Glu Arg Leu Leu Trp, Leu Tyr Arg 425 430 GTC CTG GCC CAC ATG GAG GCC ACG Val Leu Ala His Met Glu Ala Thr 445 AGG CCC CTT TCC Arg Pro Leu Ser GGG GTG CCC CTG GAC GTG GCC Gly Val Arg Leu Asp Val Ala 450 455 TAT CTC AGG GCC T1yr Leu Arg Ala TCC CTG GAG GTG Ser Leu Clu Val GGG GAG ATC GCC Gly Glu Ile Ala CTC GAG GCC GAG Leu Glu Ala Glu TTC CGC CTG GCC Phe Arg Leu Ala 285 CAC CCC TTC AC CTC AAC TCC CGG GC His Pro Phe Asn Leu Asn Ser Arg Asp 485 CTC GAA AGG GTC Leu Glu Arg Val CTC TTT Leu Phe 495 GAC GAG CTA Asp Giu Leu CGC TCC ACC Arg Ser Thr 515 CTT CCC GCC ATC Leu Pro Ala Ile A-AG ACG GAG AAG Lys Thr Glu Lys A.CC GCC AAG Thr Gly Lys 510 GCC CAC CCC Ala His Pro AGC GCC GCC GTC Ser Ala Ala Val GAG GCC CTC CC Giu Ala Leu Arg ATC GTG Ile Val 530 GAG ZAAG P.TC CTG Giu Lys Ile Leu TAC CGG GAG CTC Tyr PArg Glu Leu AAC CTG AJ4C AGC Lys Leu Lys Ser TAC AT' CAC CCC Tyr Ile Asp Pro CCC CAC CTC ATC Pro Asp Leu Ile CAC CCC AGC P.CC CCC CC His Pro Abrg Thr Cly Arg 555 C;9( CTC C-AC ACC CC Leu H~is Thr A.rg AAC C-AG ACG GCC A~sn Gin Thr Ala CCC ACG CCC AGC Ala Thr Cly -Arg CT?. AG.T Leu Ser 575 AGC TCC C-AT Ser Ser Asp AAC CTC C-AC A-AC A~sn Leu Gin Asn CCC CTC CCC ACC Pro Val Arg Thr CCC CTT CCC Pro Leu Gly 590 CTA~ TTC CTC Leu Leu Val C-AG .A'GG ATC CCC CCC CCC TTC ATC CCC GAG C-AG CCC TCC Gin Arg Ile .tArg Arg Ala Phe Ile Ala Glu Clu Gly Trp 595 600 605 CCC CTC Ala Leu 610 CCC TAT AGC C-AC -Ala Tyr Ser Gin GAG CTC A.CG CTC Giv Leu Arg Val CCC C-AC CTC TCC Ala His Leu Ser 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 2064 2112 2160 2208 2256 CP.C GAG AAC CTG A~sp Civ Asn Leu CCC GTC TTC CCG PArg Val Phe Gin CCC CCC C-AC ATC Gly Arg Asp Ile CC C-AG ACC GCC Thr Civ Thr -Ala TCC ATC TTC GC Trp Met Phe Gly ICCC CCC C-AG CC Pro A~rg Giu -Ala CTC C-AC Val -Asp 655 CCC CTC ALTG Pro Leu Met CCC ATC TCG Cly Met Ser 675 CCC CCC CC CCC AAC JACC A.TC AAC TTC CCC CTC CTC T-AC Arg Arg -Ala Ala Lys Thr Ile Asn Phe Cly Val Leu Tyr 660 665 670 CCC C-AC CCC CTC Ala His Arg Leu C-AC GAG~. CT-A CC Gin Ciu Leu -Ala CCT TAC GAG Pro Tyr Civ GAG GCC Clu Ala 690 C-AG CCC TTC ATT Gin -Ala Phe Ile CCC TAC TI'T CAiC Arg Tyr Phe Gin TTC CCC AAG. GTG Phe Pro Lys Val CCC TCG A~TT GAGC A~'la Trp Ile Ciu ACC CTC C-AG C-AG Thr Leu Civ Ciu ACC A.GC CCC CCC A~rg A.rg Arg Gly CTC C-AG ACC CTC Val Civ Thr Leu CCC CCC CCC CC Gly Arg .Arg PArg CTC CC-A C-AC CTA Val Pro -Asp Leu GAG CC Civ -Ala 735 CCC GTC AP.G ACC CTC CCC C-AC CC CCC C-AC CCC ATC CCC TTC J..C ATC Arg Val Lys Ser Val Arg Glu Ala Ala Giu Arg Met -Ala Phe A~sn Met 740 745 750 286

CCC

Pro crC Leu

CAC

His 785

GCC

Ala

CCC

Pro

GAG

Glu (2) GTC CAG GGC ACC GCC GCC GAC CTC ATG Val Gln Gly Thr Ala Ala Asp Leu Met 755 760 TTC CCC AGG CTG GAG GAA ATG GGG GCC Phe Pro Ary Leu Glu Glu Met Gly Ala 770 775 GAC GAG CTG GTC CTC GAG GCC CCA AAAI Asp Glu Leu Val Leu Glu Ala Pro Lys 1 790 CGG CTG GCC AAG GAG GTC ATG GAG GGG Arg Leu Ala Lys Glu Val Met Glu Gly 805 810 CTG GAG GTG GAG GTG GGG ATA GGG GAG Leu Glu Val Glu Val Gly Ile Gly Glu 820 825

TGATAG

INFORMATION FOR SEQ ID NO:130: SEQUENCE CHARACTERISTICS: LENGTH: 833 amino acids TYPE: amino acid TOPOLOGY: linear 2304 2352 2400 2448 2496 2505 (ii) MOLECULE TYPE: protein .44.

(xi) SEQUENCE Asn Ser Gly Met Val Asp Gly His Leu Thr Thr Ser Lys Ser Leu Leu Val Phe Asp Ala Tyr Lys Ala Gly Ala Leu Ile Lys 100 Val Pro Gly Tyr 115 Ala Glu Lys Glu 130 Leu Tyr Gln Leu Tyr Leu Ile Thr 165 Asp Gln Trp Ala 180 DESCRIPTION: SEQ ID NO:130: Leu Pro Leu Phe Glu Pro Lys His Leu Ala Tyr Arg Thr Phe Arg Gly Glu Pro Val Gln Ala Lys Ala Leu Lys Glu Asp Gly 55 Lys Ala Pro Ser Phe Arg His Arg Ala Pro Thr Pro Glu Asp Glu Leu Val Asp Leu Leu Gly 105 Glu Ala Asp Asp Val Leu Ala 120 Gly Tyr Glu Val Arg Ile Leu 135 140 Leu Ser Asp Arg Ile His Val 150 155 Pro Ala Trp Leu Trp, Glu Lys 170 Asp Tyr Arg Ala Leu Thr Gly *4 *4 4.

287 Asn Leu Pro Gly Val Lys Gly Asn eu Po Gy Va ysGl Ile Giv Giu Lys Thr, la. Ar, L. s e 15200 205 Z).

Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg 210 215 220 Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu 252330 235 240 Lys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu 245 250 255 Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala 260 265 270 Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280 285 Leu Giu Ser Pro Lys Ala Leu Giu Giu Ala Pro Trp Pro Pro Pro Giu 290 295 300 Gly Ala Phe Vai Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala 305 310 315 320 Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala 325 330 335 Pro Giu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Giu Ala Arg Gly Leu 340 345 350 Leu Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365 Pro Pro Gly Asp Asp Pro Met Leu Leu Ala-Tyr Leu Leu Asp Pro Ser 370 375 380 Asn Thr Thr Pro Giu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr 385 390 395 400 Giu Giu Ala Gly Giu Arg Ala Ala Leu Ser Giu Arg Leu Phe Ala Asn 405 410 415 Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg 420 425 430 Giu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Giu Ala Thr 435 440 445 Gl.. y Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Giu Val *-450 455 460 Ala Gly Giu Ile Ala Arg Leu Giu Ala Glu Val Phe Arg Leu Ala Gly 465 470 475 480 .His Pro Phe Asn Leu Asn Ser Arg Asp Gin Leu Glu Arg Val Leu Phe *485 490 495 Asp Giu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 500 505 510 .Arg Ser Thr Ser Ala Ala Val Leu Giu Ala Leu Arg Glu Ala His Pro 515 520 525 Ile Val Giu Lys Ile Leu Gin Tyr Arg Giu Leu Thr Lys Leu Lys Ser 530 535 540 .Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg 545 550 555 560 28 Leu His Thr Arg LeuHisThrArgPhe Asn Gin Thr Ala Ala Thr Gly Arg Leu Ser 57S Ser Ser Gin Arg Ala Leu 610 Gly Asp 625 Thr Glu Pro Leu Gly Met Glu Ala 690 Arg Ala 705 Val Giu Arg Vai Pro Val Leu Phe 770 His Asp 785 Ala Arg Pro Leu Glu Pro 580 Arg Tyr Asn Ala Ara 660 Ala Ala Ile Le u Ser 740 Gly Arg Leu Ala Val 820 Asn Ile Pro 585 Ile Ala Giu Giu Leu Arg Val Phe Gin Phe Gly Val 650 Lys Thr Ile 665 Ser Gin Glu 680 Arg Tyr Phe Leu Giu Glu Arg Arg Tyr 730 Ala Ala Giu 745 Asp Leu Met 760 Met Gly Ala Ala Pro Lys Met Giu Giy 810 Ile Gly Giu 825 Thr Pro 590 Trp Leu bUS Ala His Arg Asp Glu Ala Gly Val 670 Ile Pro 685 Phe Pro Arg Arg Asp Leu Ala Phe 750 Ala Met 765 Leu Leu Ala Giu Pro Leu Leu Ser 830

S

9*.

INFORMATION FOR SEQ ID NO:131: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2505 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: Idesc "DNA" (ix) FEATURE: NANE/KEY: CDS LOCATION: 1. .2499 289 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: AAT TCG GGG ATG CTG CCC CTC TTT Asn Ser Gly Met Leu Pro Leu Phe CCC AAG GGC CG Pro Lys Gly Arg GTC CTC Val Leu CTG GTG GAC GGC CTC ACC Gly Leu Thr CAC CAC CTG GCC COC ACC TTC CAC Arg rnr Phe His GCC CTG AAG Ala Leu Lys ACC AGC COG GGG Thr Ser Arg Gly CCG OTO CAG GCG GTC TAC GGC TTC Pro Val Gin Ala Val Tyr Gly Phe 144 GCC AAG Ala Lys AGC CTC CTC AAG Ser Leu Leu Lys CTC AAG GAG GAC Leu Lys Giu Asp GAC GCG GTG ATC Asp Ala Val Ile GTC TTT GAC 0CC Val Phe Asp Ala 0CC CCC TCC TTC Ala Pro Ser Phe CAC GAG GCC TAC His Glu Ala Tyr GO TAC AAG OCG GGC COG GCC CCC ACO CCG GAG GAC TTT CCC COG CAA Gly Tyr Lys Ala Gly Arg Ala Pro Thr Pro Giu Asp Phe Pro Arg Gin 90 288 CTC GCC CTC Leu Aia Leu ATC AAG GAG CTG GTG GAC CTC CTG 000 CTG GCG COC CTC Ile Lys Giu Leu Val Asp Leu Leu Oly Leu Ala Arg Leu 100 105 110 GAG GTC CCG GGC TAC GAG GCG Glu Val Pro Gly Tyr Glu Ala 115 GAC GAC GTC CTG 0CC AOC CTG 0CC AAG Asp Asp Val Leu Ala Ser Leu Ala Lys 120 125 AAG GCG Lys Ala 130 GAA AAG GAG GGC Giu Lys Giu Gly GAG 0TC CGC ATC Giu Val Arg Ile ACC 0CC GAC AAA Thr Ala Asp Lys 432 CTT TAC CAG CTC Leu Tyr Gin Leu TCC GAC COC ATC Ser Asp Arg Ile GTC CTC CAC CCC Val Leu His Pro GOG TAC CTC ATC Oly Tyr Leu Ile CCG GCC TOG CTT Pro Ala Trp Leu GAA AAG TAC GGC Giu Lys Tyr Gly CTG AGO Leu Arg 175 CCC GAC CAG Pro Asp Gin 0CC GAC TAC CG Ala Asp Tyr Arg CTG ACC 000 GAC Leu Thr Oly Asp GAG TCC GAC Giu Ser Asp 190 AGO AAG CTT Arg Lys Leu AAC CTT CCC 000 GTC AAO GOC Asn Leu Pro Gly Val Lys Gly 195 000 GAG AAG ACO Oly Olu Lys Thr

OCO

Ala 205 CTG GAG Leu Giu 210 GAG TOG 000 AGC Giu Trp, Oly Ser GAA 0CC CTC CTC Glu Ala Leu Leu AAC CTG GAC CG Asn Leu Asp Arg CTG AAO CCC 0CC ATC COG GAG AAG ATC CTO 0CC CAC ATO GAC OAT CTG Leu Lys Pro Ala Ile Arg Giu Lys Ile Leu Ala His Met A.sp Asp Leu 225 230 235 240 AAG CTC TCC TG Lys Leu Ser Trp OTO GAC TTC 0CC Val Asp Phe Ala CTG 0CC AAO OTO Leu Ala Lys Val ACC GAC CTG CCC Thr Asp Leu Pro CTG GAG Leu Oiu 255 AAA AGO COO GAG Lys Arg Arg Glu GAC COG GAG AGO Asp Arg Giu Arg CTT AGO 0CC Leu Arg Ala 270 290 TTT CTG GAG AGG CTT GAG TTT GGC AGC CTC CTC CAC GAG TTC GGC CT Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Giu Phe Gly Leu 275 280 285 CTG GAA Leu Giu 290 AGC CCC AAG GCC Ser Pro Lys Ala GAG GAG GCC CCC Giu Giu Ala Pro CCC CCG CCG GAA Pro Pro Pro Giu

GGG

Gly 305 GCC TTC GTG GGC Ala Phe Val Gly GTG CTT TCC CGC Val Leu Ser Arg GAG CCC ATG TGG Glu Pro Met Trp GAT CTT CTG GCC CTG GCC GCC GCC AGG GGG GGC CGG GTC CAC CGG GCC Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala 325 330 335 CCC GAG CCT Pro Glu Pro CTC CCC AAA Leu Ala Lys 355 AAA GCC CTC AGG Lys Ala Leu Arg CTG AAG GAG GCG Leu Lys Giu Ala CGG GGG CTT Arg Gly Leu 350 C'rT GGC CTC Leu Gly Leu GAC CTG AGC GTT CTG GCC CTG AGG GAA Asp Leu Ser Val Leu Ala Leu Arg Giu CCC CCC Pro Pro 370 GGC GAC GAC CCC Gly Asp Asp Pro CTC CTC GCC TAC Leu Leu Ala Tyr CTG GAC CCT TCC Leu Asp Pro Ser ACC ACC CCC GAG Thr Thr Pro Glu

GGG

Gly 390 GTG GCC CGG CGC Val Ala Arg Arg GGC GGG GAG TGG Gly Gly Glu Trp GAG GAG GC GGG Glu Glu Ala Gly CGG GCC GCC CTT Arg Ala Ala Leu GAG AGG CTC TTC Giu Arg Leu Phe GCC AAC Ala Asn 415 CTG TGG GGS Leu Trp Gly GAG GTG GAG Giu Val Glu 435 CTT GAG GGG GAG Leu Ciu Giy Giu GAG AGG CTC CTT TGG CTT TAC CGG Giu Arg Leu Leu Trp Leu Tyr Arg 425 430 GTC CTG GCC CAC ATG GAG GCC ACG Val Leu Ala His Met Giu Ala Thr 445 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 AGG CCC CTT TCC Arg Pro Leu Ser GGG GTG CGC CTG GAC GTG GCC TAT CTC AGG GCC TTG Giy Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu 450 455 460 TCC CTG GAG GTG Ser Leu Glu Val GCC GGG GAG ATC GCC Ala Gly Giu Ile Ala 465 CTC GAG GCC GAG Leu Glu Ala Glu 2TC CGC CTG GCC Phe Arg Leu Ala CAC CCC TTC AAC His Pro Phe Asn AAC TCC CGG GAC Asn Ser Arg Asp CTG GAA AGG GTC Leu Glu Arg Val CTC TTT Leu Phe 495 GAC GAG CTA GGG CTT CCC GCC ATC GGC Asp Glu Leu Gly Leu Pro Ala Ile Gly 500 505 AAG ACG GAG AAG Lys Thr Glu Lys ACC GGC AAG Thr Gly Lys 510 CCC CAC CCC Ala His Pro CGC TCC ACC Arg Ser Thr 515 AGC GCC GCC GTC Ser Ala Ala Val GAG GCC CTC CGC Glu Ala Leu Arg ATC GTG Ile Val 530 GAG AAG ATC CTG Glu Lys Ile Leu CAG TAC CGG Gin Tyr Arg 535 GAG CTC ACC AAG CTG AAG, AGC Giu Leu Thr Lys Leu Lys Ser 540 291 ACC TAC ATT GAC CCC TTG CCG GAC CTC ATC CAC CCC AGG ACG Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr 545 550 555 GGC CGC Gly Arg 560 CTA AGT Leu Ser 575 CTC CAC ACC CGC Leu His Thr Arg AAC CAG ACG GCC Asn Gin Thr Ala GCC ACG GGC AGG Ala Thr Gly Arg AGC TCC GAT Ser Ser Asp CAG AGG ATC Gin Arg Ile 595 AAC CTC CAG AAC Asn Leu Gin Asn CCC GTC CGC ACC Pro Val Arg Thr CCG GGG Pro Leu Gly 590 CTA TTG GTG Leu Leu Val CGC CGG GCC TTC Arg Arg Ala Phe GCC GAG GAG GGG Ala Glu Glu Gly GCC CTG Ala Leu 610 GTC TAT AGC CAG Val Tyr Ser Gin GAG CTC AGG GTG Glu Leu Arg Val GCC CAC CTC TCC Ala His Leu Ser GGC GAC GAG AAC CTG ATC CGG GTC TTC CAG GAG GGG CGG GAC ATC CAC Gly Asp Glu Asn Leu lie Arg Val Phe Gin Glu Gly Arg Asp Ile His 625 630 635 640 ACG GAG ACC GCC Thr Glu Thr Ala AGC TGG ATG TTC GGC GTC CCC CGG GAG GCC GTG GAC Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp 645 650 655 CCC CTG ATG Pro Leu Met GGC ATG TCG Gly Met Ser 675 CGG GCG GCC AAG Arg Ala Ala Lys ATC AAC TTC GGG Ile Asn Phe Gly GTC CTC TAC Val Leu Tyr 670 CCT TAC GAG Pro Tyr Glu GCC CAC CGC CTC Ala His Arg Leu CAG GAG CTA GCC Gin Glu Leu Ala 1680 1728 1776 1824 1872 1920 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 GAG GCC Glu Ala 690 CAG GCC TTC ATT Gin Ala Phe ile CGC TAC TTT CAG Arg Tyr Phe Gin TTC CCC AAG GTG Phe Pro Lys Val GCC TGG ATT GAG Ala Trp lie Glu ACC CTG GAG GAG Thr Leu Glu Glu AGG AGG CGG GGG Arg Arg Arg Gly GTG GAG ACC CTC Val Glu Thr Leu GGC CGC CGC CGC Gly A-rg Arg Arg GTG CCA GAC CTA Val Pro Asp Leu GAG GCC Glu Ala 735 CGG GTG AAG Arg Val Lys CCC GTC CAG Pro Val Gin 755 GTG CGG GAG GCG Val Arg Glu Ala GAG CGC ATG GCC Glu Arg Met Ala TTC AAC ATG Phe Asn Met 750 ATG GTG AAG Met Val Lys GGC ACC GCC GCC Gly Thr Ala Ala CTC ATG AAG CTG Leu Met Lys Leu CTC TTC Leu Phe 770 CCC AGG CTG GAG Pro Arg Leu Glu ATG GGG GCC AGG Met Gly Ala Arg CTC CTT CAG GTC Leu Leu Gln Val GAC GAG CTG GTC Asp Glu Leu Val GAG GCC CCA AAA Glu Ala Pro Lys AGG GCG GAG GCC Arg Ala Glu Ala GCC CGG CTG GCC Ala Arg Leu Ala GAG GTC ATG GAG Glu Val Met Glu GTG TAT CCC CTG Val Tyr Pro Leu GCC GTG Ala Val 815 -292 CCC CTG GAG GTG GAG GTG GGG ATA GGG GAG GAC TGG CTC TCC GCC AAG Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 GAG TGATAG Glu 1FRu±i10N.~r tux S.Q ID NO:i32: SEQUENCE CHARACTERISTICS: LENGTH: 833 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 2496 2505 (xi) SEQUENCE Asn Ser Gly Met Val Asp Gly His Leu Thr Thr Ser Lys Ser Leu Leu Val Phe Asp Ala Tyr Lys Ala Gly Ala Leu Ile Lys 100 Val Pro Gly Tyr 115 Ala Giu Lys Giu 130 Leu Tyr Gin Leu Tyr Leu Ile Thr 165 Asp Gin Trp Ala 180 Leu Pro Gly Val 195 Glu Glu Trp Gly 210 Lys Pro Ala Ile Leu Ser Trp Asp 245 Asp Phe Ala Lys 260 DESCRIPTION: SEQ ID NO:132: Leu Pro Leu Phe Giu Pro Lys His Leu Ala Tyr Arg Thr Phe Arg Gly Giu Pro Val Gin Ala Lys Ala Leu Lys Glu Asp Gly 55 Lys Ala Pro Ser Phe Arg His Arg Ala Pro Thr Pro Glu Asp Glu Leu Val Asp Leu Leu Gly 105 Giu Ala Asp Asp Val Leu Ala 120 Gly Tyr Glu Val Arg Ile Leu 135 140 Leu Ser Asp Arg Ile His Val 150 155 Pro Ala Trp, Leu Trp Glu Lys 170 Asp Tyr Arg Ala Leu Thr Gly 185 Lys Gly Ile Gly Giu Lys Thr 200 Ser Leu Glu Ala Leu Leu Lys 215 220 Arg Glu Lys Ile Leu Ala His 230 235 Leu Ala Lys Val Arg Thr Asp 250 Arg Arg Glu Pro Asp Arg Glu

S.

Sees

S

S

Se S S

S

.55.

9* 5.

5 5

S

0S *5 *0S6@5

S

S. *S S S

S

55 0 5*

S..

293 Phe Leu Glu Arg Leu Glu Phe 275 Ser Leu Leu His Glu Phe Gly Leu 285 Leu Glu Ser Pro 0* S S S. S

*SSS

S

*SS.

tS..S.

*SS. S

S

*5 S. S

S

S.

Glu Glu Ala Leu Ser Arq Ala Arg Gly 330 Arg Asp Leu 345 Leu Ala Leu 360 Leu Leu Ala Ala Arg Arg Ala Leu Ser 410 Glu Glu Arg 425 Ala Val Leu 440 Tyr Leu Arg Glu Ala Glu Arg Asp Gln 490 Ile Gly Lys 505 Leu Glu Ala 520 Tyr Arg (flu Asp Leu Ile Thr Ala Thr 570 Asn Ile Pro 585 Ile Ala Glu 600 Glu Leu Arg Val Phe Gln Asp Glu Asn Leu Ile Arg 630 294 Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp 645 650 655 Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr 660 665 670 Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu 660 685 Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val 690 695 700 Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr 705 710 715 720 Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735 Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750 Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760 765 Leu Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val 770 775 780 His Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val 785 790 795 800 Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val 805 810 815 Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 SGlu INFORMATION FOR SEQ ID NO:133: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: AAAATTCCTT TCTCTTTGCC CTTTGCTTCC INFORMATION FOR SEQ ID NO:134: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(l..2, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxycytosine -295 (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(3, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxythymidine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: ici~izoui~fference LOCATION: replace(4..5, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxyadenosine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(6..8, OTHER INFORMATION: /note= "The residues at these positions are a 2'deoxythymidine 5'-0-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(9, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxyguanosine 5'-O-(1-Thiomonophosphate)." (ix) FEATURE: NAME/KEY: misc difference LOCATION: replace(10, OTHER INFORMATION: /note= "The residue at this position is a 2'deoxycytosine 5'-O-(1-Thiomonophosphate)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: CCTAATTTGC CAGTTACAAA ATAAACAGCC C 31 INFORMATION FOR SEQ ID NO:135: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: o oTGTGGAATTG TGAGCGG 17 INFORMATION FOR SEQ ID NO:136: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: TGGAGGCTCT CCATCAAAAA C 21 -296 INFORMATION FOR SEQ ID NO:137: SEQUENCE CHARACTERISTICS: LENGTH: 296 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137: TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGA GTGCAGTTTG GTGATTTTAT TCCAAAAAAT ATTATCTCCT TTGAAGATTT AAAAGGGAAA AAAGTAGCTA 120 TTGATGGAAT GAATGCATTA TATCAGTTTT TAACATCTAT ACGTTTGAGA GATGGTTCTC 180 CATTGAGAAA TAGAAAAGGA GAGATAACCT CAGCATATAA CGGAGTTTTT TATAAAACCA 240 TACATTTGTT AGAGAATGAT ATAACTCCAA TCTGGGTTTT TGATGGAGAG CCTCCA 296 INFORMATION FOR SEQ ID NO:138: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" S(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: TAATCTGTAT CAGGCTG 17 INFORMATION FOR SEQ ID NO:139: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: GTTTTTGATG GAGAGCCTCC A 21 INFORMATION FOR SEQ ID NO:140: SEQUENCE CHARACTERISTICS: LENGTH: 889 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: GTTTTTGATG GAGAGCCTCC AGAATTCAAA AAGAAAGAGC TCGAAAAAAG AAGAGAAGCG AGAGAGGAAG CTGAAGAAAA GTGGAGAGAA GCACTTGAAA AAGGAGAGAT AGAGGAAGCA 120 -297

AGAAAATATG

CTCTTAGAGC

GCATATATGG

CTTTTTGGAG

GGGAAAAATG

GAATTAAAGC

AACCCAGGAG

AA.AGATCCGC

TTCTTCCTAA

GAGGGAATAC

GGATTAGAGA

TTCAAGAGAT

CTTGGCTGTT

CCCAAAGAGC

TTATGGGAAT

CCGCAAAGGG

CTCCAAGACT

TCTACGTCGA

TAACAAGAGA

GAATAAkAGGG

TAGCAAAGTT

ACCCACCAGT

TA.AAGTTCTT

GGCTTAAGAA

AACCTTAAAG

TTGGCGGATG

AACCAGGGTA

TCCTATAGT

GAGCGTGTAT

TGTTAGAAAC

GATAAAGCCC

AAAGCTCATT

CATAGGCCTT

CCAAAAGCAA

CACAGATAAC

ATGTGACGAG

GGCAATCAAA

TCTATTGCAA

AATGAAATGC

CAAGCACCTA

GCATCGGCTA

TTAACAATA.A

GAGTTGATAA

GAACTAGCAA

AAAAAAGCTT

AGCGATGTGG

TACAAT'rTAG

CATGACTTTA

AGTGGAAAAC

TG7TATACTG

TCATCGAGGA

GCGAGGGAGA

GTCAAGATTA

CAGGAAAAAG

TTTTGGAGGA

TCCTCGTTGG

TAGAGATTGT

ATTTATATGC

TGTGGAGAGA

GTGAGGAAAG

AATCAACCCT

ACGCGCTGCA

TGCAAAAAAA

GGCCCAAGCT

CGATTCCCTA

A.AAGTTGCCT

AGTACTCAAG

AACAGACTAC

TAGACACTCA

AATAAAAGAG

TCCCGACGA.A

AGTAAAGAAT

TGAAAGTTGG

GGCATGCAAG

AGAGAAGATT TTCAGCCTGA TACAGATTA INFORMATION FOR SEQ ID NO:l4l: SEQUENCE CHARACTERISTICS: LENGTH: 1164 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGA GTGCAG TTT G GTGATTTTAT TCCAAAAAAT TTGATGGAAT GAATGCATTA CATTGAGAAA TAGAAAAGGA TACATTTGTT AGAGAATGAT TCAAAAAGAA AGAGCTCGAA GAGA.AGCACT TGAAAAAGGA GGGTAAATGA AATGCTCATC TAGTTCAAGC ACCTAGCGAG TGTATGCATC GGCTAGTCAA GAAACTTAAC AATAACAGGA AGCCCGAGTT GATAATTTTG TCATTGAACT AGCAATCCTC GCCTTAAAAA AGCTTTAGAG AGCAA.AGCGA TGTGGATTTA ATAACTACA.A TTI'AGTGTGG

ATTATCTCCT

TATCAGTTTT

GAGATAACCT

ATAACTCCAA

AAAAGAAGAG

GAGATAGAGG

GAGGATGCAA

GGAGAGGCCC

GATTACGATT

AAAAGAAAGT

GAGGAAGTAC

GTTGGAACAG

ATTGTTAGAC

TATGCAATAA

AGAGATCCCG

TTGAAGATTT

TAACATCTAT

CAGCATATAA

TCTGGGTTTT

AAGCGAGAGA

AAGCAAGAAA

AAAAACTCTT

AAGCTGCATA

CCCTACT'IrT

TGCCTGGGAA

TCAAGGAATT

ACTACAACCC

ACTCAAAAGA

AAGAGTTCTT

ACGAAGAGGG

AAAAGGGA.AA

ACGTTTGAGA

CGGAGTTT1T

TGATGGAGAG

GGAAGCTGAA

ATATGCCCAA

AGAGCTTATG

TATGGCCGCA

TGGAGCTCCA

AAATGTCTAC

AAAGCTAACA

AGGAGGAATA

TCCGCTAGCA

CCTAAACCCA

AATACTAAAG

AAAGTAGCTA

GATGGTTCTC

TATAAAACCA

CCTCCAGAAT

GAAAAGTGGA

AGAGCAACCA

GGAATTCCTA

AAGGGGAGCG

AGACTTGTTA

GTCGAGATAA

AGAGAAAAGC

AAGGGCATAG

AAGTTCCAAA

CCAGTCACAG

TTCTTATGTG

298 ACGAGCATGA CTTTAGTGAG GAAAGAGTAA AGAATGGATT AGAGAGGCTT AAGAAGGCAA TCAAAAGTGG AAAACAATCA ACCCTTGAAA GTTGGTTCAA GAGATAACCT TAAAGTCTAT TGCAATGTTA TACTGACGCG CTGCAGGCAT GCAAGCTTGG CTGTTTTGGC GGATGAGAGA AGATTTTCAG CCTGATACAG ATTA INFORMATION FOR SEQ ID NO:142: SEQUENCE CHARACTERISTICS: LENGTH: 296 base pairs TYPE: nucleic acid STRAflDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142: TGTGGAA7TTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGT GTCCCA-ATTG GTGAGATTAT ACCAAGAAA GAAATTGAGT TAGAAAACCT ATACGGGAAA AAAATCGCAA TCGACGCTCT TAATGCAATC TACCAATTTT TGTCCACAAT AAGACAGAAA GATGGAACTC CACTTATGGA TTCA-AAGGGT AGAATAACCT CCCACCTAAG CGGGCTCTTT TACAGGACAA TAAACCTAAT GGAGGCTGGA ATAAAACCTG TGTATGTTTT TGATGGAGAG CCTCCA INFORMATION FOR SEQ ID NO:143: SEQUENCE CHARACTERISTICS: LENGTH: 840 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143: GTTTTTGATG GAGAGCCTCC AAAGTTAAAG GAGAAAACAA GGAAAGTTAG GAGAGAGATG 1020 1080 1140 1164 120 180 240 296

AAAGAGAAAG

GCTAAGTATG

TTGTTAAGTT

AGCTATATGG

TTATATGGAG

ATTGAATTAA

GCCATATTTA

GCTTATGAAT

TACGATGAGA

CTAAAATTGC

TATGATAGGG

CTGAACTTAA

CAAAGAGGGT

TGATGGGCAT

CAAAGAAGGG

CTCCGAGAGT

ATGAGGTTTT

TGGGAACTGA

TGGTTAGAAG

TTAAGAGGAT

CAGATAAAGA

TTAAAAAGCA

GATGAAAGAG

TAGCTATCTA

TCCGTATGTT

AGATGTTGG

TGTTAGAAAT

AGAGGATTTA

CTATAATCCA

TGGTGTAGCT

ATTTAAAGAG

GCAATTAAAA

ACTCCGAAAA

GAAGCTCCCT

GCAGTTGTAA

TTAACAACTA

AGAAT'rrCTT

GGAGGAGTTA

AAGGATGTTT

CCAAAGGTTA

AGGAGGATTT

TGGTTGAAAA

CTGAGGGAGA

GTCAAGATTA

CAAAGGAGAT

TGGATGATTT

AAGGAATAGG

TGAAAAAAGA

CCGATAACTA

TTGATGAAAA

TAATTGCAAA

TGAAGAAGCT

CTGCAA.ATAT

GGCACAAGCA

TGATGCCTTG

GCCAGAACTT

GATAGATATA

ATTTAAAAGG

GGTTGAATAC

TTCATTAAGC

TGACTTAAT

CAAAACTAAG

GGGAATTATA AAATTCTTAG TGTTGATAAA CTCTATAACT 299 CA.AAAAACAT TAGATGCATG GTTTAAATAA TTTATATAAT TTTGTGGGAT GTCGACCTGC AGGCATGCAA GCTTGGCTGT TTTGGCGGAT GAGAGAAGAT TTTCAGCCTG ATACAGATTA INFORMATION FOR SEQ ID NO:144: SEQUENCE CHARACTERISTICS: T.Wr~iu r !;SEpj- TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc ',DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144: TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGT 00

GTGAGATTAT

TCGACGCTCT

CACTTATGGA

TAAACCTAAT

TAAAGGAGAA

AAGAGGCAAT1

ATCTAACTCC

ATGTTGAAGC

TTTGGGCAGT

GAAATTTAAC

ATTTAAGAAT

ATCCAGGAGG

TAGCTAAGGA

AAGAGCCAAA

TTATAAAATT

ATAAACTCTA

AATAATTTAT

ACCAAGAAAA

TAATGCAATC

TTCAAAGGGT

GGAGGCTGGA

AACAAGGAAA

TAAAAAGGAG

GAAAATGGTT

TCCCTCTGAG

TGTAAGTCAA

AACTACAAAG

TTCTTTGGAT

AGTTAAAGGA

TGTTTTGAA.A

GGTTACCGAT

CTTAGTTGAT

TAACTTAATT

ATAA7rTGT

GAAATTGAGT

TACCAATTTT

AGAATAACCT

ATAA.AACCTG

GTTAGGAGAG

GATTTTGAAG

GAAAACTGCA

GGAGAGGCAC

GATTATGATG

GAGATGCCAG

GATTTGATAG

ATAGGATTTA

AAAGAGGTTG

AACTATTCAT

GAAAATGACT

GCAAACAAAA

GGGATGTCGA

TAGAAAACCT

TGTCCACAAT

CCCACCTAAG

TGTATGTTTT

AGATGAAAGA

AAGCTGCTAA

AATATT-TGTT

AAGCAAGCTA

CCTTGTTATA

AACTTATTGA

ATATAGCCAT

AAAGGGCTTA

AATACTACGA

TAAGCCTAAA

TTAATTATGA

CTAAGCAAAA

CCTGCAGGCA

ATACGGGAAA

AAGACAGAAA

CGGGCTCTTT

TGATGGAGAG

GAAAGCTGAA

GTATGCAAAG

AAGTTTGATG

TATGGCAAAG

TGGAGCTCCG

ATTAAATGAG

ATTTATGGGA

TGAATTGGTT

TGAGATTAAG

ATTGCCAGAT

TAGGGTTAAA

AACATTAGAT

TGCAAGCTTG

GTCCCAATTG

AAAATCGCA-A

GATGGAACTC

TACAGGACAA

CCTCCAAAGT

CTTAAGATGA

AGGGTTAGCT

GGCATTCCGT

AAGGGAGATG

AGAGTTGTTA

GTTTTAGAGG

ACTGACTATA

AGAAGTGGTG

AGGATATTTA

AAAGAGGGAA

A.AGCATGTTG

GCATGGTTTA

GCTGTTTTGG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1115 CGGATGAGAG AAGATTTTCA GCCTGATACA GATTA INFORMATION FOR SEQ ID NO:145: SEQUENCE CHARACTERISTICS: LENGTH: 386 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" -300 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145: TGTGGA.ATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGT GTGAGATTAT ACCAAGAAAA GAAATTGAGT TAGAAAACCT ATACGGGAAA TCGACGCTCT TAATGCAATC TACCAATTTT TGTCCACAAT AAGACAGAAA CACTTATGGA TTCAAAGGGT AGAATAACCT CCCACCTAAG CGGGCTCTTT TAAACCTAAT GGAGGCTGGA ATAAAACCTG TGTATGTTTT TGATGGAGAA TCAAAAAGAA AGAGCTCGAA AAAAGAAGAG AAGCGAGAGA GGAAGCTGAA GAGAAGCACT TGAAAAAGGA GAGATA INFORMATION FOR SEQ ID NO:146: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146: TACTTAGCAG CTTCTTCTAT CTCTCCTTTT TCA INFORMATION FOR SEQ ID NO:147: SEQUENCE CHARACTERISTICS: LENGTH: 668 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION:. /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:

GTCCCAATTG

AAAATCGCAA

GATGGAACTC

TACAGGACAA

CCTCCAGAAT

GAAAAGTGGA

GAAGAAGCTG CTAAGTATGC AAAGAGGGTT

TGCAAATATT

GCACAAGCAA

GATGCCTTGT

CCAGAACTTA

ATAGATATAG

TTTAAAAGGG

GTTGAATACT

TCATTAAGCC

GACTTTAATT

AAAACTAAGC

CAGCGGTA

TGTTAAGTTT

GCTATATGGC

TATATGGAGC

TTGAATTAAA

CCATATTTAT

CTTATGAATT

ACGATGAGAT

TAAAATTGCC

ATGATAGGGT

AAAAAACATT

GATGGGCATT

AAAGAAGGGA

TCCGAGAGTT

TGAGGTTTTA

GGGAACTGAC

GGTTAGAAGT

TAAGAGGATA

AGATAAAGAG

TAAAAAGCAT

AGATGCATGG

AGCTATCTAA

CCGTATGTTG

GATGTTTGGG

GTTAGAAATT

GAGGATTTAA

TATAATCCAG

GGTGTAGCTA

TTTAAAGAGC

GGAATTATAA

GTTGATAAAC

TTTAAACACC

CTCCGAAAAT

AAGCTCCCTC

CAGTTGTAAG

TAACAACTAC

GAATTTCTTT

GAGGAGTTAA

AGGATGTTTT

CAAAGGTTAC

AATTCTTAGT

TCTATAACTT

ACCACCACCA

GGTTGAAAAC

TGAGGGAGAG

TCAAGATTAT

AAAGGAGATG

GGATGATTTG

AGGAATAGGA

GAAAAAAGAG

CGATAACTAT

TGATGAAAAT

AATTGCAAAC

CCACTAACTG

301 INFORMATION FOR SEQ ID NO:14B: SEQUENCE CHARACTERISTICS: LENGTH: 53 base pairs TYPE: nucleic acid STRANDEDNESS: single TOP(OT.rOr.: I"~ (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc ',DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148: TACCGCTGCA GTTAGTGGTG GTGGTGGTGG TGTTTAAACC ATGCATCTA.A TGT INFORMATION FOR SEQ ID NO:149: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149: GAAGAAGCTG CTAAGTA INFORMATION FOR SEQ ID NO:150: SEQUENCE CHARACTERISTICS: LENGTH: 1054 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc ",DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150: TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGT GTCCCAATTG GTGAGATTAT ACCAAGAAAA GAAATTGAGT

TCGACGCTCT

CACTTATGGA

TAAACCTAAT

TCAAAAAGAA

GAGAAGCACT

ATCTAACTCC

ATGTTGA.AGC

TTGGGCAGT

GAAATTTAAC

ATTTAAGAAT

A77:CAGGAGG

TAGCTAAGGA

TAATGCAATC

TTCAAAGGGT

GGAGGCTGGA

AGAGCTCGAA

TGAAAAAGGA

GAAAATGGTT

TCCCTCTGAG

TGTAAGTCAA

AACTACAAAG

TTCTTTGGAT

AGTTAAAGGA

TGTTTTGA.AA

TACCAATTTT

AGAATAACCT

ATAAAACCTG

AAAAGAAGAG

GAGATAGAAG

GAAAACTGCA

GGAGAGGCAC

GATTATGATG

GAGATGCCAG

GATTTGATAG

ATAGGATTTA

AAAGAGGTTG

TAGAAAACCT ATACGGGAAA TGTCCACAAT AAGACAGAAA CCCACCTAAG CGGGCTCTTT TGTATGTTTT TGATGGAGAA AAGCGAGAGA GGAAGCTGAA A.AGCTGCTA.A GTATGCAAAG AATATTTGTT AAGTTI'GATG AAGCAAGCTA TATGGCAAAG CCTTGTTATA TGGAGCTCCG AACTTATTGA ATTAAATGAG

AAAATCGCAA

GATGGAACTC

TACAGGACAA

CCTCCAGAAT

GAAAAGTGGA

AGGGTTAGCT

GGCATTCCGT

AAGGGAGATG

AGAGTTGTTA

GTT TTAGAGG

ATATAGCCAT

AAAGGGCTTA

AATACTACGA

ATTATGGGA ACTGACTATA TGAATTGGTT AGAAGTGGTG TGAGATTAAG AGGATATTTA 302 AAGAGCCAAA GGTTACCGAT AACTATTCAT TAAGCCTAAA ATTGCCAGAT AAAGAGGGAA TTATAAAATT CTTAGTTGAT GAAAATGACT TTAATTATGA TAGGGTTAAA AAGCATGTTG ATAAACTCTA TAACTTAATT GCAAACAAAA CTAAGCAAAA AACATTAGAT GCATGGTTTA AACACCACCA CCACCACCAC TAACTGCAGC GGTA INFORMATION FOR SEQ ID NO:151: SEQUENCE CHARACTERISTICS: LENGTH: 514 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151: 900 960 1020 TGTGGAATTG TGAGCGGATA

GTGATTTTAT

TTGATGGAAT

CATTGAGAAA

TACATTTGTT

TAAAGGAGAA

AAGAGGCAAT

ATCTAACTCC

ATGTTGAAGC

TCCAAAAAAT

GAATGCATTA

TAGAAAAGGA

AGAGAATGAT

AACAAGGAAA

TAAAAAGGAG

GAAAATGGTT

TCCCTCTGAG

ACAATTTCAC

ATTATCTCCT

TATCAGTTTT

GAGATAACCT

ATAACTCCAA

GTTAGGAGAG

GATTTTGAAG

GAAAACTGCA

ACAGGAAACA GACCATGGGA TTGAAGATTT AAAAGGGAAA TAACATCTAT ACGTTTGAGA CAGCATATAA CGGAGTTTTT TCTGGGTTTT TGATGGTGAG AGATGAAAGA GAAAGCTGAA AAGCTGCTAA GTATGCAAAG AATATTTGTT AAGTTTGATG

GTGCAGTTTG

AAAGTAGCTA

GATGGTTCTC

TATAAAACCA

CCACCAAAGT

CTTAAGATGA

AGGGTTAGCT

GGCATTCCGT

r r r r r r GGAGAGGCCC AAGC INFORMATION FOR SEQ ID NO:152: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152: GCTTGGGCCT CTCCCTC INFORMATION FOR SEQ ID NO:153: SEQUENCE CHARACTERISTICS: LENGTH: 667 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" 303 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153: GAGGGAGAGG CCCAAGCTGC ATATATGGCC GCAAAGGGGA

CAAGATTACG

GGAAAAAGAA

TTGGAGGAAG

CTCGTTGGAA.

GAGATTGTTA

TTATATGCAA

TGGAGAGATC

GAGGAAAGAG

TCAACCCTTG

GCGCTGCAGG

CAGATTA

ATTCCCTACT

AGTTGCCTGG

TACTCAAGGA

CAGACTACAA

GACACTCAAA

TAAALAGAGTT

CCGACGAAGA

TAAAGAATGG

AAAGTTGGTT

CATGCAAGCT

TTTTGGAGCT

GAAAAATGTC

ATTAAAGCTA

CCCAGGAGGA

AGATCCGCTA

CTTCCTAAAC

GGGAATACTA

ATTAGAGAGG

CAAGAGATAA

TGGCTGTTTT

CCAAGACTTG

TACGTCGAGA

ACAAGAGAAA

ATAAAGGGCA

GCAAAGTTCC

CCACCAGTCA

AAGTTCTTAT

CTTAAGAAGG

CCTTAAAGTC

GGCGGATGAG

GCGTGTATGC

TTAGAAACTT

TAAAGCCCGA

AGCTCATTGA

TAGGCC TTAA

AAAAGCAAAG

CAGATAACTA

GTGACGAGCA

CAATCAAAAG

TATTGCAATG

AGAAGATTTT

ATCGGCTAGT

AACAATAACA

GTTGATAATT

ACTAGCAATC

AAAAGCTTTA

CGATGTGGAT

CAATTTAGTG

TGACTTTAGT

TGGAAAACAA

TTATACTGAC

CAGCCTGATA

g INFORMATION FOR SEQ ID NO:154: Wi SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154: GAGCGAGAGG CCCAAGC INFORMATION FOR SEQ ID NO:155: SEQUENCE CHARACTERISTICS: LENGTH: 1164 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155: TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGA GTGATTTTAT TCCAAAAAAT ATTATCTCCT TTGAAGATTT AAAAGGGAAA TTGATGGAAT GAATGCATTA TATCAGTTTT TAACATCTAT ACGTTTGAGA CATTGAGAAA TAGAAAAGGA GAGATAACCT CAGCATATAA CGGAGTTTTT TACATTTGTT AGAGAATGAT ATAACTCCAA TCTGGGTTTT TGATGGTGAG TAAAGGAGAA AACAAGGAAA GTTAGGAGAG AGATGAAAGA GAAAGCTGAA AAGAGGCAAT TAAAAAGGAG GATTTTGAAG AAGCTGCTAA GTATGCAAAG ATCTAACTCC GAAAATGGTT GAAAACTGCA AATATTTGTT AAGTTTGATG

GTGCAGTTTG

AAAGTAGCTA

GATGGTTCTC

TATAAAACCA

CCACCAAAGT

CTTAAGATGA

AGGGTTAGCT

GGCATTCCGT

304

ATGTTGAAGC

TGTATGCATC

GAAACTTAAC

AGCCCGAGTT

TCATTGAACT

GCCTTAAA.AA

AGCAAAGCGA

ATAACTACAA

ACGAGCATGA

TCAAAAGTGG

TGCAATGTTA

AGAT=YCAG

TCCC'rCTGAG

GGCTAGTCAA

AATAACAGGA

GATAATTTTG

AGCAATCCTC

AGCTTTAGAG

TGTGGATTTA

TT TAGTGTGG

CTTTAGTGAG

AAAACAATCA

TACTGACGCG

CCTGATACAG

GGAGAGGCCC

GATTACGATT

AAAAGAAAGT

GAGGAAGTAC

GTTGGAACAG

ATTGTTAGAC

TATGCAATAA

AGAGATCCCG

GAA.AGAGTAA

ACCCTTGAAA

CTGCAGGCAT

ATTA

AAGCTGCATA

CCCTACTTTT

TGCCTGGGAA

TCAAGGAATT

ACTACAACCC

ACTCAAAAGA

AAGAGTTCTT

ACGAAGAGGG

AGAATGGATT

GTTGGTTCA-A

GCAAGCTTGG

TATGGCCGCA

TGGAGCTCCA

AAATGTCTAC

AAAGCTAACA

AGGAGGAATA

TCCGCTAGCA

CCTAAACCCA

AATACTAAAG

AGAGAGGCTT

GAGATAACCT

CTGTTTTGGC

AAGGGGAGCG

AGACTTGTTA

GTCGAGATAA

AGAGAAAAGC

AAGGGCATAG

AAGTTCCAAA

CCAGTCACAG

TTCTTATGTG

AAGAAGGCAA

TAAAGTCTAT

GGATGAGAGA

540 600 660 720 780 840 900 960 1020 1080 1140 1164 INFORMATION FOR SEQ ID NO:156: Wi SEQUENCE CHARACTERISTICS: LENGTH: 514 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156: TGTGGAArrc TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGGGT GTCCCAATTG a

GTGAGATTAT

TCGACGCTCT

CACTTATGGA

TAAACCTAAT

TCAAAAAGAA

GAGAAGCACT

GGGTAAATGA

ACCAAGAAAA

TAATGCAATC

TTCAAAGGGT

GGAGGCTGGA

AGAGCTCGAA

TGAAAAAGGA

AATGCTCATC

GAAATTGAGT

TACCAATTTT

AGAATAACCT

ATAAAACCTG

AAAAGAAGAG

GAGATAGAGG

GAGGATGCAA

GGAGAGGCCC

TAGAAAACCT ATACGGGAAA TGTCCACAAT AAGACAGAAA CCCACCTAAG CGGGCTCTITT TGTATGTTTT TGATGGAGAA AAGCGAGAGA GGAAGCTGAA AAGCAAGAAA ATATGCCCAA AAAAACTCTT AGAGCTTATG

AAGC

AAAATCGCAA

GATGGAACTC

TACAGGACAA

CCTCCAGAAT

GAAAAGTGGA

AGAGCAACCA

GGAATTCCTA

TAGTTCAAGC ACCTAGCGAG a a a INFORMATION FOR SEQ ID NO:157: SEQUENCE CHARACTERISTICS: LENGTH: 618 base pairs TYPE: nucleic acid STRA1NDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA,- 305 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1SY: GAGGGAGAGG CCCAAGCAAG

CAAGATTATG

AAGGAGATGC

GATGATTGA

GGAATAGGAT

AAAAAAGAGG

GATAACTATT

GATGAAAATG

ATTGCAAACA

TGTGGGATGT

TCAGCCTGAT

ATGCCTTGTT

CAGAACTTAT

TAGATATAGC

TTAAAAGGGC

TTGAATACTA

CATTAAGCCT

ACTTTAATTA

AAACTAAGCA

CGACCTGCAG

ACAGATTA

CTATATGGCA

ATATGGAGCT

TGAATTAAAT

CATAT'rATG

TTATGAATTG

CGATGAGATT

AAAATTGCCA

TGATAGGGTT

AAAAACATTA

GCATGCAAGC

AAGAAGGGAG

CCGAGAGTrG

GAGGTTTTAG

GGAACTGACT

GTTAGAAGTG

AAGAGGATAT

GATAAAGAGG

AAAAAGCATG

GATGCATGGT

TTGGCTGTTT

ATGTTTGGGC

TTAGAAATTT

AGGATTTAAG

ATAATCCAGG

GTGTAGCTAA

TTAAAGAGCC

GAATTATAAA

TTGATAAACT

TTAAATAATT

TGGCGGATGA

AGTTGTAAGT

AACAACTACA

AATTTCTTTG

AGGAGTTAAA

GGATGTTT TG

AAAGGTTACC

ATTCTTAGTT

CTATAACTTA

TATATAATTT

GAGAAGATTT

INFORMATION FOR SEQ ID NO:158: SEQUENCE CHARACTERISTICS: LENGTH: 1115 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158: TGTGGA.ATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA

GTGAGATTAT

TCGACGCTCT

CACTTATGGA

TAAACCTAAT

TCAAAAAGA.

GAGAAGCACT

GGGTAAATGA

TAGTTCAAGC

TTTGGGCAGT

GAAATTTAAC

ATTTAAGAAT

ATCCAGGAGG

TAGCTAAGGA

AAGAGCCAAA

TTATAAAATT

ATAAACTCTA

ACCAAGAAAA

TAATGCAATC

TTCAAAGGGT

GGAGGCTGGA

AGAGCTCGAA

TGAAAAAGGA

AATGCTCATC

ACCTAGCGAG

TGTA.AGTCAA

AACTACAAAG

TTCTTTGGAT

AGTTAAAGGA

TGTTTTGAAA

GGTTACCGAT

CTTAGTTGAT

TAACTTAATT

GAAATTGAGT

TACCAATTT

AGAATAACCT

ATAAAACCTG

AAAAGAAGAG

GAGATAGAGG

GAGGATGCAA

GGAGAGGCCC

GATTATGATG

GAGATGCCAG

GATTGATAG

ATAGGATTTA

AAAGAGGTTG

AACTATTCAT

GAAAATGACT

GCAAACAAAA

TAGAAAACCT

TGTCCACAAT

CCCACCTAAG

TGTATGTTTT

AAGCGAGAGA

AAGCAAGAAA

AAAAACTCTT

AAGCAAGCTA

CCTTGTTATA

AACTTATTGA

ATATAGCCAT

AAAGGGCTTA

AATACTACGA

TAAGCCTAAA

TTAATTATGA

CTAAGCAAAA

GACCATGGGT

ATACGGGAAA

AAGACAGAAA

CGGGCTCTT

TGATGGAGAA

GGAAGCTGAA

ATATGCCCAA

AGAGCTTATG

TATGGCAAAG

TGGAGCTCCG

ATTAAATGAG

ATTTATGGGA

TGAATTGGTT

TGAGATTAAG

ATTGCCAGAT

TAGGGTTAAA

AACATTAGAT

GTCCCAATTG

AAAATCGCAA

GATGGAACTC

TACAGGACAA

CCTCCAGAAT

GAAAAGTGGA

AGAGCAACCA

GGAATTCCTA

AAGGGAGATG

AGAGTTGTTA

GTTTTAGAGG

ACTGACTATA

AGAAGTGGTG,

AGGATATTTA

AAAGAGGGAA

A.AGCATGTTG

GCATGGTTTA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 306 AATAATTTAT ATAATT= GT GGGATGTCGA CCTGCAGGCA TGCAAGCTTG GCTGTT= GG CGGATGAGAG AAGAT=TTCA GCCTGATACA GATTA INFORMATION FOR SEQ ID NO:159: SEQUENCE CHARACTERISTICS: LENCTH: 2505 L abt pairs TYPE: nucleic acid STRANflEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159: 1080 1115 ATGGAGGCGA TGCTTCCGCT

CACCTGGCCT

GTGCAGGCGG

AAGGCCGTCT

GCCTACAAGG

AAGGAGCTGG

GACGTTCTCG

ACCGCCGACC

GGCCACCTCA

GTGGACTTCC

GGGGAGAAGA

AACCTGGACC

CTCAGGCTCT

GCCCAGGGGC

GGCAGCCTCC

TGGCCCCCGC

GCGGAGCTTA

TTGGCGGGGC

TTGGCCTCGA

CTCCTGGACC

ACGGAGGACG

CGCCTCGAGG

CGGGTCCTGG

CTTTCCCTGG

GGCCACCCCT

AGGCTTCCCG

CTGGAGGCCC

ACCGCACCTT

TCTACGGCTT

TCGTGGTCTT

CGGGGAGGGC

TGGACCTCCT

CCACCCTGGC

GCGACCTCTA

TCACCCCGGA

GCGCCCTCGT

CCGCCCTCAA

GGGTAAAGCC

CCTTGGAGCT

GGGAGCCCGA

TCCACGAGTT

CGGAAGGGGC

AAGCCC TGGC

TAAAGGACCT

GGGAGGGGCT

CCTCCAACAC

CCGCCCACCG

GGGAGGAGAA

CCCACATGGA

AGCTTGCGGA

TCAACCTCAA

CCTTGGGGAA

TACGGGAGGC

CTTTGAACCC

CTTCGCCCTG

CGCCAAGAGC

TGACGCCAAG

CCCGACCCCC

GGGGTTTACC

CAAGAAGGCG

CCAACTCGTC

GTGGCTTTGG

GGGGGACCCC

GCTCCTCAAG

AGAAAACGTC

CTCCCGGGTG

CCGGGAGGGG

CGGCCTCCTG

CTTCGTGGGC

CGCCTGCAGG

CAAGGAGGTC

AGACCTCGTG

CACCCCCGAG

GGCCCTCCTC

GCTCCTTTGG

GGCCACCGGG

GGAGATCCGC

CTCCCGGGAC

GACGCAAAAG

CCACCCCATC

AAAGGCCGGG

AAGGGCCTCA

CTCCTCAAGG

GCCCCCTCCT

GAGGACTTCC

CGCCTCGAGG

GAAAAGGAGG

TCCGACCGCG

GAGAAGTACG

TCCGACAACC

GAGTGGGGAA

CGGAGAAGA

CGCACCGACC

CTTAGGGCCT

GAGGCCCCCG

TTCGTCCTCT

GACGGCCGGG

CGGGGCCTCC

CCCGGGGACG

GGGGTGGCGC

TCGGAGAGGC

CTCTACCACG

GTACGGCGGG

CGCCTCGAGG

CAGCTGGAAA

ACAGGCAAGC

GTGGAGAAGA

TCCTCCTGGT

CCACGAGCCG

CCCTGAAGGA

TCCGCCACGA

CCCGGCAGCT

TCCCCGGCTA

GGTACGAGGT

TCGCCGTCCT

GCCTCAGGCC

TCCCCGGGGT

GCCTGGAAAA

TCAAGGCCCA

TCCCCCTGGA

TCCTGGAGAG

CCCCCCTGGA

CCCGCCCCGA

TGCACCGGGC

TCGCCAAGGA

ACCCCATGCT

GGCGCTACGG

TCCATCGGAA

AGGTGGAAAA

ACGTGGCCTA

AGGAGGTCTT

GGGTGCTCTT

GCTCCACCAG

TCCTCCAGCA

GGACGGCCAC

GGGCGAACCG

GGACGGGTAC

GGCCTACGAG

CGCCCTCATC

CGAGGCGGAC

GCGCATCCTC

CCACCCCGAG

GGAGCAGTGG

CAAGGGCATC

CCTCCTCAAG

CCTGGAAGAC

GGTGGACCTC

GCTGGAGTTC

GGAGGCCCCC

GCCCATGTGG

AGCAGACCCC

CCTCGCCGTC

CCTCGCCTAC

GGGGGAGTGG

CCTCCTTAAG

GCCCCTCTCC

C=TCAGGCC

CCGCTTGGCG

TGACGAGCTT

CGCCGCGGTG

CCGGGAGCTC

120 180 240 300 360 420 480 540 600 660 720 780 640 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 307

ACCAAGCTCA

CGCCTCCACA

CCCAACCTGC

GTGGCCGAGG

CTCGCCCACC

CACACCCAGA

CGCCGGGCGG

TCCCAGGAGC

AGCTTCCCCA

TACGTGGAAA

AGCGTCAGGG

GACCTCATGA

ATGCTCCTCC

GTGGCGGCTT

AGAACACCTA

CCCGCTTCAA

AGAACATCCC

CGGGTTGGGC

TCTCCGGGGA

CCGCAAGCTG

CCAAGACGGT

TTGCCATCCC

AGGTGCGGGC

CCCTCTTCGG

AGGCCGCGGA

AGCTCGCCAT

AGGTCCACGA

TGGCCAAGGA

CGTGGACCCC

CCAGACGGCC

CGTCCGCACC

GTTGGTGGCC

CGAAAACCTG

GATGTTCGGC

GAACTTCGGC

CTACGAGGAG

CTGGATAGAA

AAGAAGGCGC

GCGCATGGCC

GGTGAAGCTC

CGAGCTCCTC

GGCCATGGAG

CTCCCAAGCC

ACGGCCACGG

CCCTTGGGCC

CTGGACTATA

ATCAGGGTCT

GTCCCCCCGG

GTCCTCTACG

GCGGTGGCCT

AAGACCCTGG

TACGTGCCCG

TrCAACATGC

TTCCCCCGCC

CTGGAGGCCC

AAGGCCTATC

TCCGCCAAGG

TCGTCCACCC

GGAGGCTTAG

AGAGGATCCG

GCCAGATAGA

TCCAGGAGGG

AGGCCGTGGA

GCATGTCCGC

TTATAGAGCG

AGGAGGGGAG

ACCTCAACGC

CCGTCCAGGG

TCCGGGAGAT

CCCAAGCGCG

CCCTCGCCGT

GTTAG

GAGGACGGGC

TAGCTCCGAC

CCGGGCCTTC

GCTCCGCGTC

GAAGGACATC

CCCCCTGATG

CCATAGGCTC

CTACTTCCAA

GAAGCGGGGC

CCGGGTGAAG

CACCGCCGCC

GGGGGCCCGC

GGCCGAGGAG

GCCCCTGGAG

1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2505 GTGGAGGTGG GGATGGGGGA GGACTGGCTT 9 *9 INFORMATION FOR SEQ ID NO:160: Wi SEQUENCE CHARACTERISTICS: LENGTH: 834 amino acids TYPE: amino acid STRANDEDNESS; not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160: Met Glu Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

C

C.

C.

Val Asp Gly Leu Thr Thr His Leu Ala Tyr Thr Phe Phe Ala Leu Lys Gly Tyr Gly Phe Ala Ser Arg Gly Glu Val Gln Ala Val Lys Ser Val Val Leu Leu Lys Ala Phe Asp Ala Lys 70 Lys Glu Asp Gly Lys Ala Val Phe Pro Ser Phe Arg His Glu Asp Glu Ala Tyr Phe Pro Arg Tyr Lys Ala Gly Arg Ala Pro Thr 85 Leu Ala Leu Glu Val Pro 115 Lys Ala Glu 130 Lys Glu Leu Val Tyr Glu Ala Asp Leu Leu Gly Phe Thr Arg Leu 105 110 Asp Val Leu Ala Thr Leu Ala Lys 125 Val Arg Ile Leu Thr Ala Asp Arg 140 Lys Glu Gly 308 Asp Leu Tyr Gin Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145 150 155 160 Gly His Leu Ile Thr Pro Glu Trp Leu Trp Giu Lys Tyr Gly Leu Arg 165 170 175 Pro Glu Gin Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 1ifl I a5 190 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu 195 200 205 Leu Lys Glu Trp, Gly Ser Leu Giu Asn Leu Leu Lys Asn Leu Asp Az-g 210 215 220 Val Lys Pro Giu Asn Val Arg Giu Lys Ile Lys Ala His Leu Glu Asp 225 230 235 240 Leu Arg Leu Ser Leu Giu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu 245 250 255 Glu Val Asp Leu Ala Gin Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg 260 265 270 Ala Phe Leu Glu Arg Leu Glu Phe Giy Ser Leu Leu His Glu Phe Gly 275 280 285 Leu Leu Glu Ala Pro Ala Pro Leu Giu Glu Ala Pro Trp, Pro Pro Pro 290 295 300 Giu Gly Ala Phe Vai Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp 305 310 315 320 Ala Glu Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg 325 330 335 Ala Ala Asp Pro Leu Aia Gly Leu Lys Asp Leu Lys Glu Val Arg Gly .340 345 350 9*-Leu Leu Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp .355 360 365 0.*Leu Val Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375 380 Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Giu Trp 385 390 395 400 V..Thr Glu Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg 405 410 415 Asn Leu Leu Lys Arg Leu Giu Gly Glu Giu Lys Leu Leu Trp Leu Tyr 9420 425 430 His Giu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala *435 440 445 Thr Gly Val Arg Arg Asp Val Ala Tyr Leu Gin Ala Leu Ser Leu Glu .450 455 460 *Leu Ala Giu Giu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala *465 470 475 480 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gin Leu Giu Arg Val Leu 485 490 495 Phe Asp Glu Leu Arg Leu Pro Ala Leu Giy Lys Thr Gin Lys Thr Gly 500 505 510 -309 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His 515 520 525 Pro Ile Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys 530 535 540 Asn Thr Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly 545 155 Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu 565 570 575 Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu 580 585 590 Gly Gin Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605 Val Ala Leu Asp Tyr Ser Gin Ile Glu Leu Arg Val Leu Ala His Leu 610 615 620 Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gin Giu Gly Lys Asp Ile 625 630 635 640 His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val 645 650 655 Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670 Tyr Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala Ile Pro Tyr 675 680 685 Glu Giu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gin Ser Phe Pro Lys 690 695 700 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Giu Gly Arg Lys Arg Gly 0:0:705 710 715 720 9960 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn ~725 730 735 see* Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn *740 745 750 met Pro Val Gin Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val 755 760 765 *Lys Leu Phe Pro Arg Leu Arg Giu Met Gly Ala Arg Met Leu Leu Gin 770 775 780 Val His Asp Glu Leu Leu Leu Giu Ala Pro Gin Ala Arg Ala Glu Giu 0*0:785 790 795 800 Val Ala Ala Leu Ala Lys Giu Ala Met Giu Lys Ala Tyr Pro Leu Ala 5805 810 815 Val Pro Leu Glu Val Giu Val Gly Met Gly Glu Asp Trp Leu Ser Ala *820 825 830 *Lys Gly INFORMATION FOR SEQ ID NO:161: SEQUENCE CHARA CTE RISTICS: LENGTH: 2511 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 310 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: Idesc DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161: ATGAATTCCG AGGCGATGCT TCCGCTCTTT GAACCCAAAG GGCCACCArr(

GAACCGGTGC

GGGTACAAGG

TACGAGGCCT

CTCATCAAGG

GCGGACGACG

ATCCTCACCG

CCCGAGGGCC

CAGTGGGTGG

GGCATCGGGG

CTCAAGAACC

GAAGACCTCA

GACCTCG CCC

GAGTTCGGCA

GCCCCCTGGC

ATGTGGGCGG

GACCCCTTGG

GCCGTCTTGG

GCCTACCTCC

GAGTGGACGG

CTTAAGCGCC

CTCTCCCGGG

CAGGCCCTTT

TTGGCGGGCC

GAGCTTAGGC

GCGGTGCTGG

GAGCTCACCA

ACGGGCCGCC

TCCGACCCCA

GCCTTCGTGG

CGCGTCCTCG

GACATCCACA

T(nnrCeT~ cr(

AGGCGGTCTA

CCGTCTTCGT

ACAAGGCGGG

AGCTGGTGGA

TTCTCGCCAC

CCGACCGCGA

ACCTCATCAC

ACTTCCGCGC

AGAAGACCGC

TGGACCGGGT

GGCTCTCCTT

AGGGGCGGGA

GCCTCCTCCA

CCCCGCCGGA

AGCTTAAAGC

CGGGGCTAAA

CCTCGAGGGA

TGGACCCCTC

AGGACGCCGC

TCGAGGGGGA

TCCTGGCCCA

CCCTGGAGCT

ACCCCTTCAA

TTCCCGCCTT

AGGCCCTACG

AGCTCAAGAA

TCCACACCCG

ACCTGCAGAA

CCGAGGCGGG

CCCACCTCTC

CCCAGACCGC

f,.hfri ,T r~lrf

CGGCTTCGCC

GGTCTTTGAC

GAGGGCCCCG

CCTCCTGGGG

CCTGGCCA-AG

CCTCTACCAA

CCCGGAGTGG

CCTCGTGGGG

CCTCAAGCTC

AAAGCCAGAA

GGAGCTCTCC

GCCCGACCGG

CGAGTTCGGC

AGGGGCCTTC

CCTGGCCGCC

GGACCTCAAG

GGGGCTAGAC

CAACACCACC

CCACCGGGCC

GGAGAAGCTC

CATGGAGGCC

TGCGGAGGAG

CCTCAACTCC

GGGGAAGACG

GGAGGCCCAC

CACCTACGTG

CTTCAACCAG

CATCCCCGTC

TTGGGCGTTG

CGGGGACGAA

AAGCTGGATG

AAGAGCCTCC

GCCAAGGCCC

ACCCCCGAGG

TTTACCCGCC

AAGGCGGAAA

CTCGTCTCCG

CTTTGGGAGA

GACCCCTCCG

CTCAAGGAGT

AACGTCCGGG

CGGGTGCGCA

GAGGGGCTTA

CTCCTGGAGG

GTGGGCTTCG

TGCAGGGACG

GAGGTCCGGG

CTCGTGCCCG

CCCGAGGGGG

CTCCTCTCGG

CTTTGGCTCT

ACCGGGGTAC

ATCCGCCGCC

CGGG.ACCAGC

CAAAAGACAG

CCCATCGTGG

GACCCCCTCC

ACGGCCACGG

CGCACCCCCI2

GTGGCCCTGG

GCCGGGTCCT

TCAAGGCCCT

CCTCCTTCCG

ACTTCCCCCG

TCGAGGTCCC

AGGAGGGGTA

ACCGCGTCGC

AGTACGCCCT

ACAACCTCCC

GGGGAAGCCT

AGAAGATCAA

CCGACCTCCC

GGGCCTTCCT

CCCCCGCCCC

TCCTCTCCCG

GCCGGGTGCA

GCCTCCTCGC

GGGACGACCC

TGGCGCGGCG

AGAGGCTCCA

ACCACGAGGT

GGCGGGACGT

TCGAGGAGGA

TGGAAAGGGT

GCAAGCGCTC

AGAAGATCCT

CAAGCCTCGT

CCACGGGGAG

TGGGCCAGAG

ACTATAGCCA

CCTGGTGGAC

GAAGGAGGAC

CCACGAGGCC

GCAGCTCGCC

CGGCTACGAG

CGAGGTGCGC

CGTCCTCCAC

CAGGCCGGAG

CGGGGTCAAG

GGAAAACCTC

GGCCCACCTG

CCTGGAGGTG

GGAGAGGCTG

CCTGGAGGAG

CCCCGAGCCC

CCGGGCAGCA

CAAGGACCTC

CATGCTCCTC

CTACGGGGGG

TCGGAACCTC

GGAAAAGCCC

GGCCTACCTT

GGTCTTCCGC

GCTCTTTGAC

CACCAGCGCC

CCAGCACCGG

CCACCCGAGG

GCTTAGTAGC

GATCCGCCGG

GATAGAGCTC

GGAGGGGAAG

CGTGGACCCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 ft.

ft ft. ft 4 ft ft ft ft ft ft ft.

AACCTGATCA GGGTCTTCCA TTCGGCGTCC CCCCGGAGGC 311

CTGATGCGCC

AGGCTCTCCC

TTCCAAAGCT

CGGGGCTACG

GTGAAGAGCG

GCCGCCGACC

GCCCGCATGC

GAGGAGGTGG

GGGCGGCCAA

AGGAGCT TGC

TCCCCAAGGT

TGGAAACCCT

TCAGGGAGGC

TCATGAAGCT

TCCTCCAGGT

CGGCTTTGGC

GACGGTGAAC

CATCCCCTAC

GCGGGCCTGG

CTTCGGA.AGA

CGCGGAGCGC

CGCCATGGTG

CCACGACGAG

CAAGGAGGCC

TTCGGCGTCC

GAGGAGGCGG

ATAGAAAAGA

AGGCGCTACG

ATGGCCTTCA

AAGCTCTTCC

CTCCTCCTGG

ATGGAGAAGG

TGGCTT'rCCG

TCTACGGCAT

TGGCCTTTAT

CCCTGGAGGA

TGCCCGACCT

ACATGCCCGT

CCCGCCTCCG

AGGCCCCCCA

CCTATCCCCT

CCAAGGGTTA

GTCCGCCCAT

AGAGCGCTAC

GGGGAGGAAG

CAACGCCCGG

CCAGGGCACC

GGAGATGGGG

AGCGCGGGCC

CGCCGTGCCC

G

2040 2100 2160 2220 2280 2340 2400 2460 2511 CTGGAGGTGG AGGTGGGGAT GGGGGAGGAC INFORMATION FOR SEQ ID NO:162: SEQUENCE CHJARACTERISTICS: LENGTH: 836 amino acids TYPE: amino acid STR.ANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162: Met Asn Ser Glu Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val Lys Gly Leu Gly His His Leu Ala Thr Ser Arg Gly Glu 40 Tyr Arg Thr Phe Phe Ala Val Phe Lys Ser Leu Leu Val Val Phe Asp Lys Ala Leu 55 Ala Lys Ala Pro Val Gin Lys Glu Asp Pro Ser Phe Pne Ala Leu Val Tyr Gly Tyr Lys Ala Arg His Glu Tyr Glu Ala Tyr Lys Ala Gly Arg Ala Pro 90 Thr Pro GlU Asp Phe Pro Arg Gin Leu Arg Leu Glu 115 Ala Lys Lys 130 Leu Ile Lys Pro Gly Tyr Glu Leu 105 Glu Ala 120 Gly Tyr Val Ser Val Asp Leu Leu Ala Glu Lys Asp Asp Val Glu Val Arg 140 Asp Arg Val Gly Phe Thr 110 Ala Thr Leu Leu Thr Ala Asp 145 Pro Arg Asp Leu Tyr Glu Gly His Leu 165 Ala Val Leu Thr Pro Glu Trp Glu Lys Tyr Gly 175 Leu Arg Pro Ser Asp Asn 195 Gin Trp Val Pro Gly Val Asp Phe 185 Lys Gly 200 Arg Ala Leu Val 190 Thr Ala Leu Ile Gly Glu 312 Asp 225 Glu Pro Leu Phe Pro 305 Met His Arg Leu Asp 385 Glu His Leu Glu Leu 465 Leu Val1 Thr Ala Leu 545 Thr 55

SS

*SS.

S

*SSS.S

S. 55

S

55 313 Arg Leu Ser Ser Ser Asp Pro Asn Leu Gin Asn Ile Pro Val Arg Thr 580 585 590 Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp 595 600 605 Ala Leu Val Ala Leu As Ty er Gn le Clu Lcu Arg Val Leu Ala 610 615 620 His Leu Ser Gly Asp Glu Asn Leu Ile Arg Val Phe Gin Glu Gly Lys 625 630 635 640 Asp Ile His Thr Gin Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu 645 650 655 Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly 660 665 670 Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gin Glu Leu Ala Ile 675 680 685 Pro Tyr Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gin Ser Phe 690 695 700 Pro Lys Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys 705 710 715 720 Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp 725 730 735 Leu Asn Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala 740 745 750 Phe Asn Met Pro Val Gin Gly Thr Ala Ala Asp Leu Met Lys Leu Ala 755 760 765 Met Val Lys Leu Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu 770 775 780 Leu Gln Val His Asp Glu Leu Leu Leu Glu Ala Pro Gin Ala Arg Ala 785 790 795 800 Glu Glu Val Ala Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro 805 810 815 Leu Ala Val Pro Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu 820 825 830 Ser Ala Lys Gly 835 INFORMATION FOR SEQ ID NO:163: SEQUENCE CHARACTERISTICS: LENGTH: 2511 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163: ATGAATTCCG AGGCGATGCT TCCGCTCTTT GAACCCAAAG GCCGGGTCCT CCTGGTGGAC GGCCACCACC TGGCCTACCG CACCTTCTTC GCCCTGAAGG GCCTCACCAC GAGCCGGGGC 120 GAACCGGTGC AGGCGGTCTA CGGCTTCGCC AAGAGCCTCC TCAAGGCCCT GAAGGAGGAC 180 **314 314 J3 £tt.TfL 3%3,G

PCGAGGCCT

rCATCAAGG

CGGACGACG

TCCTCACCG

CCGAGGGCC

AGTGGGTGG

GCATCGGGG

TCAAGAACC

*AAGACCTCA

ACCTCGCCC

AGTTCGGCA

ICCCCCTGGC

LTGTGGGCGG

;ACCCCTTGG

;CCGTCTTGG

'CCTACCTCC

3AGTGGACGG

"TTAAGCGCC

:"TCTCCCGGG

:AGGCCCTTT

rTGGCGGGCC

:;AGCTTAGGC

GCGGTGCTGG

GAGCTCACCA

ACGGGCCGCC

TCCGACCCCA

GCCTTCGTGG

CGCGTCCTCG

GACATCCACA

CTGATGCGCC

AGGCTCTCCC

TTCCAAAGCT

CGGGGCTACG

GTGAAGAGCG

ACAAGGCGGG GAGGGCCCCG

AGCTGGTGGA

TTCTCGCCAC

CCGACCGCGA

ACCTCATCAC

ACTTCCGCGC

AGAAGACCGC

TGGACCGGGT

GGCTCTCCTT

AGGGGCGGGA

GCCTCCTCCA

CCCCGCCGGA

AGCTTAAAGC

CGGGGCTAAA

CCTCGAGGGA

TGGACCCCTC

AGGACGCCGC

TCGAGGGGGA

TCCTGGCCCA

CCCTGGAGCT

ACCCCTTCAA

TTCCCGCCTT

AGGCCCTACG

AGC1TCAAGAA

TCCACACCCG

ACCTGCAGAA

CCGAGGCGGG

CCCACCTCTC

CCCAGACCGC

GGGCGGCCAA

AGGAGCTTGC

TCCCCAAGGT

TGGAAACCCT

TCAGGGAGGC

CCTCCTGGGG

CCTGGCCAAG

CCTCTACCAA

CCCGGAGTGG

CCTCGTGGGG

CCTCAAGCTC

AAAGCCAGAA

GGAGCTCTCC

GCCCGACCGG

CGAGTTCGGC

AGGGGCCTTC

CCTGGCCGCC

GGACCTCAAG

GGGGCTAGAC

CAACACCACC

CCACCGGGCC

GGAGAAGCTC

CATGGAGGCC

TGCGGAGGAG

CCTCAACTCC

GGGGAAGACG

GGAGGCCCAC

CACCTACGTG

CTTCAACCAG

CATCCCCGTC

TTGGGCGTTG

CGGGGACGAA

AAGCTGGATG

GACGGTGAAC

CATCCCCTAC

GCGGGCCTGG

CTTCGGAAGA

CGCGGAGCGC

UC:CAAGGCCC

ACCCCCGAGG

TTTACCCGCC

AAGGCGGAAA

CTCGTCTCCG

CTTTGGGAGA

GACCCCTCCG

CTCAAGGAGT

AACGTCCGGG

CGGGTGCGCA

GAGGGGCTA

CTCCTGGAGG

GTGGGCTTCG

TGCAGGGACG

GAGGTCCGGG

CTCGTGCCCG

CCCGAGGGGG

CTCCTCTCGG

CTTTGGCTCT

ACCGGGGTAC

ATCCGCCGCC

CGGGACCAC

CAAAAGACAG

CCCATCGTGG

GACCCCCTCC

ACGGCCACGG

CGCACCCCCT

GTGGCCCTGG

AACCTGATCA

TTCGGCGTCC

TTCGGCGTCC

GAGGAGGCGG

ATAGAAAAGA

AGGCGCTACG

ATGGCCTTCA

CCTCCTTCCG

ACTTCCCCCG

TCGAGGTCCC

AGGAGGGGTA

ACCGCGTCGC

AGTACGGCCT

ACAACCTCCC

GGGGAAGCCT

AGAAGATCAA

CCGACCTCCC

GGGCCT'TCCT

CCCCCGCCCC

TCCTCTCCCG

GCCGGGTGCA

GCCTCCTCGC

GGGACGACCC

TGGCGCGGCG

AGAGGCTCCA

ACCACGAGGT

GGCGGGACGT

TCGAGGAGGA

TGGAAAGGGT

GCAAGCGCTC

AGAAGATCCT

CAAGCCTCGT

CCACGGGGAG

TGGGCCAGAG

ACTATAGCCA

GGGTCTTCCA

CCCCGGAGGC

TCTACGGCAT

TGGCCTTTAT

CCCTGGAGGA

TGCCCGACCT

ACATGCCCGT

CCACGAGGCC

GCAGCTCGCC

CGGCTACGAG

CGAGGTGCGC

CGTCCTCCAC

CAGGCCGGAG

CGGGGTCAAG

GGAAAACCTC

GGCCCACCTG

CCTGGAGGTG

GGAGAGGCTG

CCTGGAGGAG

CCCCGAGCCC

CCGGGCZAGCA

CAAGGACCTC

CATGCTCCTC

CTACGGGGGG

TCGGAACCTC

GGAAAAGCCC

GGCCTACCTT

GGTCTTCCGC

GCTCTTTGAC

CACCAGCGCC

CCAGCACCGG

CCACCCGAGG

GCTTAGTAGC

GATCCGCCGG

GATAGAGCTC

GGAGGGGAAG

CGTGGACCCC

GTCCGCCCAT

AGAGCGCTAC

GGGGAGGAAG

CAACGCCCGG

CCAGGGCACC

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 315 GCCGCCGACC TCATGAAGCT CGCCATGGTG AAGCT CTT CC CCCGCCTCCG GGAGATGGGG 2340 GCCCGCATGC TCC TCCAGGT CCACAACGAG CTCCTCCTGG AGGCCCCCCA AGCGCGGGCC 2400 GAGGAGGTGG CGGCTTTGGC CAAGGAGGCC ATGGAGAAGG CCTATCCCCT CGCCGTGCCC 2460 CTGGAGGTGG AGGTGGGGAT INFORMATION FOR SEQ ID NO:164: SEQUENCE CHARACTERISTICS: LENGTH: 836 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164: Met Asn Ser Glu Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val 1 5 10 Leu Leu Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu 25 Lys Gly Leu Thr Thr Ser Arg Gly Giu Pro Val Gin Ala Val Tyr Gly 40 Phe Ala Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala 55 Val Phe Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Giu Ala 70 75 s0 Tyr Glu Ala Tyr Lys Ala Gly Arg Ala Pro Thr Pro Giu Asp Phe Pro 90 Arg Gln Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr 100 105 110 Arg Leu Giu Val Pro Gly Tyr Giu Ala Asp Asp Val Leu Ala Thr Leu 115 120 125 Ala Lys Lys Ala Glu Lys Glu Gly Tyr Giu Val Arg Ile Leu Thr Ala *130 135 140 *Asp Arg Asp Leu Tyr Gin Leu Val Ser Asp Arg Val Ala Val Leu His 145 150 155 160 Pro Glu Gly His Leu Ile Thr Pro Glu Trp Leu Trp Giu Lys Tyr Gly *165 170 175 Leu Arg Pro Giu Gin Trp, Val Asp Phe Arg Ala Leu Val Gly Asp Pro 180 185 190 Ser Asp Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu *195 200 205 Lys Leu Leu Lys Giu Trp Gly Ser Leu Giu Asn Leu Leu Lys Asn Leu 210 215 220 Asp Arg Val Lys Pro Giu Asn Val Arg Giu Lys Ile Lys Al1a His Leu *225 230 235 240 *Glu Asp Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu 245 250 255 **Pro Leu Glu Val Asp Leu Ala Gin Giy Arg Glu Pro Asp Arg Giu Gly *260 265 270 316 Leu Phe Pro 305 Met His Arg Leu Asp 385 Glu His Leu Glu Leu 465 Leu Val Thr Al a Leu 545 Thr Arg Pro Ala His 625 Ala Phe 275 Leu Leu Giu Gly Ala Giu Ala Ala 340 Leu Leu 355 Leu Val Ser Asn Thr Giu Asn Leu 420 His Giu 435 Thr Gly Leu Ala Gly His Phe Asp 500 Lys Arg 515 Pro Ile Asn Thr Arg Leu Ser Ser 580 Gly Gin 595 Val Ala Ser Gly Arg Leu 280 Pro Ala 295 Val Gly Ala Leu Leu Ala Asp Leu 360 Asp Asp 375 Pro Giu Ala His Arg Leu Lys Pro 440 Arg Asp 455 Ile Arg Asn Leu Arg Leu Ser Ala 520 Lys Ile 535 Asp Pro Arg Phe Pro Asn Arg Arg 600 Tyr Ser 615 Asn Leu Ser Giu 300 Ser Arg Asp Ala Leu 380 Arg Leu Glu Val Leu 460 Giu Asp Gly Giu Arg 540 Leu Ala Ile Ala Leu 620 Phe

*CC*.C

C.

317 Asp Ile His Thr Ala Val Asp Pro 660 Gin Thr Ala Ser Trp, Met 645 650 Leu Met Arg Arg Ala Ala Phe Gly Val Lys Thr Val Pro Pro Glu 655 Asn Phe Gly 670 Lau Atla ile Gin Ser Phe Val Leu Tvr rly Mat co bl 675 Pro Tyr Glu Glu Ala Val Ala 690 695 Pro Lys Val Arg Ala Trp Ile W4.

680 Phe Glu Ile Glu Arg Tyr 700 Lys Thr Leu Glu 715 Gly Arg Arg Arg 710 Glu Thr Glu Gly Arg Tyr Val Pro 735 Gly Tyr Val Leu Phe 730 Glu Ala Leu Asn Ala Phe Asn Met 755 Met Val Lys 770 Lys Ser Val Ala Glu Arg Met Ala 750 Val Gin Gly Ala Asp Leu Lys Leu Ala Arg Met Leu Leu Phe Pro Leu Arg Glu Met Gin Val His Asn Leu Leu Glu Gin Ala Arg Glu Glu Val Ala Ala Leu Ala Lys Glu Ala 805 810 Glu Lys Ala Leu Ala Val Pro Leu Glu 820 Val Glu Val Gly 825 Met Gly Glu Asp Trp Leu 830 a a a.

a Ser Ala Lys Gly 835 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 350 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /deac "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165: AGAGTTTGAT CATGGCTCAG ATTGAACGCT GGCGGCAGGC CTAACACATG CAAGTCGAAC GGTAACAGGA AGAAGCTTGC TTCTTTGCTG ACGAGTGGCG GACGGGTGAG TAATGTCTGG GAAACTGCCT GATGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATAACGTCG CAAGACCAAA GAGGGGGACC TTCGGGCCTC T1'GCCATCGG ATGTGCCCAG ATGGGATTAG CTAGTAGGTG GGGTAACGGC TCACCTAGGC GACGATCCCT AGCTGGTCTG, AGAGGATOAC CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG 318 .F.ORZ IDj.N ;166; o L 00: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166: kCGAATTCC GAGGCGATGC TTCCGCTC 28 2) INFORMATION FOR SEQ ID NO:167: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167: CGACGTCGA CTAACCCTTG GCGGAAAGCC 2) INFORMATION FOR SEQ ID NO:168: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168: ;CATCGCCTC GGAATTCATG GTC 23 INFORMATION FOR SEQ ID NO:169: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169: CAGGAGGAGC TCGTTGTGGA CCTGGA 26 INFORMATION FOR SEQ ID NO:170: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" 319- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170: CCGTCAACAT TTACCATGGG TGCGGA 26 INFORMATION FOR SEQ ID NO:171: SEQUENCE CrHarATETSTCS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171: CCGCCACCTC GTAGTCGACA TCCTTTTCGT G 31 INFORMATION FOR SEQ ID NO:172: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172: GGGTGTTCCC ATGGGAGTTA AACTCAGG 28 INFORMATION FOR SEQ ID NO:173: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173: CTGAATTCTG CAGAAAAAGG GG 22 INFORMATION FOR SEQ ID NO:174: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174: AGAGTTTGAT CCTGGCTCAG a.

320 INFORMATION FOR SEQ IL) NO:175: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175: ;CTGCCTC CCGTAGGAGT INFORMATION FOR SEQ ID NO:176: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176: rTCGCTGT CTCGCTGAAA GCGAGACAGC GTTT SINFORMATION FOR SEQ ID NO:177: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177: TTCGCTGT CTCGCTGAAA GCGAGACAGC GAAAGACGCT CGTGAAACGA GCGTCTTTG )INFORMATION FOR SEQ ID NO:178: SEQUENCE CHARACTERISTICS: LENGTH: 1011 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178: 'GGGTGCGG ATATTGGTGA CCTCTTTGAG AGGGAAGAGG TCGAGCTTGA GTACTTCTCA IAAAGAAAA TTGCCGTTGA TGCTTTCAAC ACGCTATACC AGTTCATCTC GATAATAAGG LGCCTGACG GTACGCCGTT AAAGGACTCA CAGGGCAGAA TCACCTCTCA CCTTTCCGGA 'CCTATACA GAGTCTCCAA CATGGTCGAG GTGGGAATCA GGCCGGTGTT TGTATTCGAC IAGAGCCAC CGGAGTTCAA GAAGGCTGAA ATTGAGGAGA GGAAAAAGAG AAGGGCTGAG :AGAGGAGA TGTGGATTGC GGCTTTGCAG GCAGGAGATA AGGACGCGAA AAAGTATGCT S *5

S

321

CAGGCTGCAG

ATGGGGATTC

GCAAAAGGCG

CCGAGACTCG

TATGTGGATG

ACGAGGGAGC

AAGGGTGTCG

GCACTCAAGG

AATCCTCCTG

ATCGAGTTCC

AAGCTCAAAG

GGAGGGTTGA

CCTTTGTCGA

ATGTGGAGTA

CCAGAAATCT

TAAAGCCGGA

AGCTCATCGA

GCGTCAAGAA

CTCTGAAAGT

TGACTGACGA

TGTGCGAGGA

CTCTGAAGTC

CGAGTACATT

TGCCCCGTCT

CACAGGAAGC

CGCAATAACG

GATAATAATT

CATAGCGATT

GGCTTTGAAC

AAATATTGAC

CTACAGAATA

GCACGACTTC

AACCCAGGCC

GTTGACTCCG

GAAGGAGAGG

CAGGATTACG

GGAAAAA(~rTh

CTGGAAAGCA

CTGGTCGGGA

TACATCAAGA

CACGTAGAGG

GAGTTCAGGG

AGCAGGGAGA

CAAAGACGCT

CGCAGGCTGC

ATTCTCTGCT

brrrw~t'n

ACCTCAAAAG

CGGACTACAA

CCTACGGAGA

AGATAAGGAA

AGCCTGACTT

GGGTCGAGAA

TTTAAGTTAC

TTACATGGCA

CTTCGGAAGC

GCTGGGTTTG

TGAGGGTGTG

TAT1'TTCAGG

TTTCTTCCTG

TGAGAAGGCC

GGCCTTGGAG

ACG CTT GAGA GGTGGTTCTG A 1011 INFORMATION FOR SEQ ID NO:179: Wi SEQUENCE CHARACTERISTICS: LENGTH: 336 amino acids TYPE: amino acid STRAN'DEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179: Met Gly Ala Asp Ile Gly Asp Leu Phe Arg Glu Glu Val Giu Leu Giu Tyr Phe Tyr Gin Phe Asp Ser Gin Gly Lys Lys Ile Asp Ala Phe Asn Thr Leu Pro Leu Lys Ser Ile Ile Gin Pro Asp Gly Thr Gly Arg Ile Ser His Leu Ser Ile Leu Tyr Arg

S

*SSS..

S

S.

S

S

so Val Ser Asn Met Val Gly Ile Arg Vai Phe Val Phe Giu Pro Pro Glu Phe Lys Lys Ala Glu Giu Arg Lys Lys Arg Arg Ala Asp Lys Asp 115 Tyr Ile Val Glu Ala Glu Glu Met 100 Ala Lys Lys Tyr Ala 120 Asp Ser Ala Lys Thr Ile Ala Ala Leu Ala Ala Gly Gin Ala Gly 110 Val Asp Glu Gly Ile Pro Leu Leu Ser 130 Phe Val Asp Ala Pro Gly Glu Ala Ala Ala Tyr Met Lys Gly Asp Giu Tyr Thr Gly Ser Gln Asp Tyr 170 Arg Leu Ala Arg Asn Leu Ala Ile 165 Asp Ser Leu 175 Thr Gly Lys Leu Phe Gly 322 s LycLC 195 Ile Ile Leu rcr Cy Lys Asa Tyr Val Asp Val Pro Giu Ile Arg Glu Gin Giu Ser Asn Lys Arg Leu Gly 210 Leu Ile Asp Ile Ala Val Gly Thr Asn Glu Giy 225 Lys Gly Val Gly Lys Ala Leu Tyr Ile Lys Thr Tyr Gly 255 Asp Ile Phe Giu Giu Ile 275 Arg Ile Giu Leu Lys Ala Vai Asn Ile Arg Asn Phe Phe Leu Asn Pro Pro Val Asp His Val 270 Asp Asp Tyr Giu Phe Leu Phe Arg Glu Asp Phe Glu Lys Giu His Asp Arg Giu Arg Lys Ala Leu Lys Leu Lys Ala Leu Lys Ser Thr Gin Ala' 325 330 2) INFORMATION FOR SEQ ID NO:180: SEQUENCE CHARACTERISTICS: LENGTH: 777 base pairs TYPE: nucleic acid STRANDEDNESS: doubie TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180: Leu Glu Arg kTGGGAGTTA. AACTCAGGGA TGTTGTATCA CCCCGCAGGA TACGCCTTGA GGACCTTAGG 3GAAGAACGG

.AGAGGGATG

tLTACTCTACA 3GAAGGTCCC rCTGAGGTTG

GCTGTAAGGT

CTTCTGGGAA

GTTAAGATGG

GCCCCAAGGG

GAACTGGAGT

CTACTCGTCG

TCGCAGTCGA

GAACACCCCT

GGACGGCCGC

ACCACCTCAA

AGTGGA-AGAG

CCTCAAGGAT

TACCCTATGT

GCGATGCATG

TTGTAAGGAA

CCACCCTCAG

GGACTGACTT

TGCAGCCAAC

CATGGATI'CC

GGTCATGGAG

GGGCGAGACC

GGCCCTTGAG

GTCCTCAGAA

ACAGGCACCC

GGCCGTGGCA

CCTCACCCTC

GGAACTCTCA

CAATGAGGGT

ACACTCTACC AGTTCCTATC

AGGGGTAGAG

AGGGAGATAA

GTGAGCAGGA

GAGGGGGACA

ATACTGGAGA

GGTGAGGGGG

TCCCAGGACT

AGCGGAAAAC

ATCAGCCACA

GTAAAGGGGA

TAACATCACA

GGGTCATATA

GGGCTGATAT

TTGACAGGGC

GTTCAAAGAG

AGGCTCAGGC

ATGACTGTCT

TTGAGGACCC

CACAGCTCGT

TAGGCGCAAG

AAGCATAAGG

CCTCAGCGGC

TGTCTTCGAT

CCGGAAGAAA

GAAAAAATAT

GC TCCTGGAA

ATCATACATG

CCTCTTTGGC

CGAGATCATT

GGATATGGCA

GAGGGGACTC

C

AAACTCATCA GGGAGAAGGG CGACA TTT TC AAAGTCATCA GGGACCTTGA AGCTTGA 777 323 INFORMATION FOR SEQ ID NO:1Bl: SEQUENCE CHARACTERISTICS: LENGTH: 258 amino acids TYPE: amino acid STRA2NDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1B1: Met Gly Val Lys Leu Arg Asp Val Val Ser Pro Arg Arg Ile Arg Leu 1 5 10 Gly Arg Ser Ser Arg Val Met Glu His Leu Ser Glu Ala Lys Glu Ser Ala Pro 150 Asp Ala 165 Ala Pro Pro Glu His Thr Glu Gly 230 Glu Lys 245 Ala Val Gin Arg His Leu Ile Arg Glu Thr Trp Lys 105 Ala Val Arg Leu Gly Glu Val Ala 170 Val Arg 185 Glu Leu Val Asp Gly Ile Ile Phe 250 Glu Ala INFORMATION FOR SEQ ID NO:182: SEQUENCE CHARACTERISTICS: LENGTH: 987 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 324 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc -'DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182: GGGAGTTA AACTCAGGGA

AAGAACGG

GAGGGATG

ACTCTACA

,AAGGTCCC

!TGAGGTTG

!TGTAAGGT

TCTGGGAA

TAAGATGG

'CCCAAGGG

ACTGGAGT

LACTCGTCG

%ACTCATCA

"TGGCGACC

~.EGATCAGGT

3CTTTTCAG,

TCGCAGTCGA

GAACACCCCT

GGACGGCCGC

ACCACCTCAA

AGTGGAAGAG

CCTCAAGGAT

TACCCTATGT

GCGATGCATG

TTGTAAGGAA

CCACCCTCAG

GGACTGACTT

GGGAGAAGGG

CCCAGGTCCT

GGAGAAAACC

AGGACCGTGT

TGTTGTATCA

TGCAGCCAAC

CATGGATTCC

GGTCATGGAG

GGGCGAGACC

GGCCCTTGAG

GTCCTCAGAA

ACAGGCACCC

GGCCGTGGCA

GGTCACCCTC

GGCCCTCTCA

CAATGAGGGT

CGACATTTTC

CAGGAGGATC

TGACGTGGAA

GAGGGATGCA

CCCCGCAGGA TACGCCTTGA ACACTCTACC AGTTCCTATC AGGGGTAGAG TAACATCACA AGGGAGATAA GGGTCATATA GTGAGCAGGA GGGCTGATAT GAGGGGGACA TTGACAGGGC ATACTGGAGA GTTCAAAGAG GGTGAGGGGG AGGCTCAGGC TCCCAGGACT ATGACTGTCT AGCGGAAAAC TTGAGGACCC ATCAGCCACA CACAGCTCGT GTAAAGGGGT ATGGCGCAAG AAAGTCATCA GGGACCTTGA TTTCTGGAGC CAGAGGTTTC GGTGTTATCG AGTTCCTGTG CTTAAAAAAT TTGAGGGTGC

GGACCTTAGG

AAGCATAAGG

CCTCAGCGGC

TGTCTTCGAT

CCGGAAGAAA

GAGAAAATAT

GCTCCTGGAA

ATCATACATG

CCTCTTTGGC

CCACATCATT

GGATATGGCA

GAGGGGACTC

AGCTGACATA

AGAGGACTAT

CACTGAACAC

ATCCTCCACC

a a a. a a a a.

AGAAGAGCC TGGAGGACTG GTTCTGA 2) INFORMATION FOR SEQ ID NO:1B3: SEQUENCE CHARACTERISTICS: LENGTH: 328 amino acids TYPE: amino acid STRANDEDNESS; not relevant TOPOLOGY: not relevant (ii) MOLE CULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183: Met Gly Val Lys Leu Arg Asp Val Val Ser Pro Arg Arg Ile Arg Leu 1 5 10 Glu Asp Leu Arg Gly Arg Thr Val Ala Val Asp Ala Ala Asn Thr Leu 20 25 Tyr Gin Phe Leu Ser Ser Ile Arg Gin Arg Asp Gly Thr Pro Leu Met 35 40 Asp Ser Arg Gly Arg Val Thr Ser His Leu Ser Gly Ile 50 55 Thr Ala Ala Val Met Glu Arg Glu Ile Arg Val Ile Tyr 65 70 Gly Arg Ser His His Leu Lys Gly Giu Thr Val Ser Arg 90 Leu Tyr Arg Val Phe Arg Ala 325 Ile Arg Lys Lys Ser Giu Val Giu Trp Lys Arg Ala Leu Giu Giu Gly 100 10510 Asp Ile Asp Arg Ala Arg Lys Tyr Ala Val Arg Ser Ser Arg Met Ser 115 120 125 Ser Giu Ile Leu Giu Ser Ser Lvs Aro Leu Leu r1ii T.pti T.aii rl T1- 130 135 140 Pro Tyr Val Gin Ala Pro Gly Giu Gly Giu Ala Gin Ala Ser Tyr Met 145 150 155 160 Val Lys Met Giy Asp Ala Trp Ala Val Ala Ser Gin Asp Tyr Asp Cys 165 170 175 Leu Leu Phe Gly Ala Pro Arg Val Val Arg Lys Val Thr Leu Ser Gly 180 185 190 Lys Leu Giu Asp Pro His Ile Ilie Giu Leu Glu Ser Thr Leu Arg Ala 195 200 205 Leu Ser Ilie Ser His Thr Gin Leu Val Asp Met Ala Leu Leu Val Gly 210 215 220 Thr Asp Phe Asn Giu Gly Val Lys Gly Tyr Gly Ala Arg Arg Gly Leu 225 230 235 240 Lys Leu Ile Arg Giu Lys Gly Asp Ile Phe Lys Val Ile Arg Asp Leu 245 250 255 Giu Ala Asp Ile Gly Gly Asp Pro Gin Val Leu Arg Arg Ile Phe Leu 260 265 270 Giu Pro Giu Val Ser Glu Asp Tyr Giu Ile Arg Trp, Arg Lys Pro Asp 275 280 285 Val Giu Gly Val Ile Giu Phe Leu Cys Thr Giu His Gly Phe Ser Giu 290 295 300 Asp Arg Val Arg Asp Ala Leu Lys Lys Phe Glu Gly Ala Ser Ser Thr 305 310 315 320 Gin Lys Ser Leu Glu Asp Trp Phe 325 INFORMATION FOR SEQ ID NO:184: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184: Met Gly Val Gin Phe Gly Asp Phe Ile Pro Lys Asn Ile Ile Ser Phe 1 5 10 Glu Asp Leu Lys Gly Lys Lys Val Ala Ile Asp Gly Met Asn Ala Leu 25 *Tyr Gin Phe Leu Thr Ser Ile Arg Leu Arg Asp Gly Ser Pro Leu Arg 40 Asn Arg Lys Gly Giu Ile Thr Ser Ala Tyr Asn Gly Val Phe Tyr Lys 55 *-326- Thr Ile His Leu Leu Glui Ag, hm Tl 'Ph, T1 'rp %Vl r~hc Asp 70 75 Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu 90 Ala Arg Glu Giu Ala Glu Glu Lys Trp Arg Giu Ala Leu Glu Lys Gly 100 105 110 Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gin Arg Ala Thr Arg Val Asn 115 120 125 Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met Gly Ile 130 135 140 Pro Ile Val Gin Ala Pro Ser Giu Gly Glu Ala Gin Ala Ala Tyr Met 145 150 155 160 Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gin Asp Tyr Asp Ser 165 170 175 Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly 180 185 190 Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Vai Glu Ile Lys Pro Glu 195 200 205 Leu Ile Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu 210 215 220 Lys Leu Ile Giu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly 225 230 235 240 Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val Arg His 245 250 255 Ser Lys Asp Pro Leu Ala Lys Phe Gin Lys Gin Ser Asp Val Asp Leu 260 265 270 @0*s 00Tyr Ala Ile Lys Giu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr *275 280 285 *Asn Leu Val Trp Arg Asp Pro Asp Giu Glu Gly Ile Leu Lys Phe Leu 290 295 300 6Cys Asp Glu His Asp Phe Ser Giu Glu Arg Val Lys Asn Gly Leu Giu *305 310 315 320 Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gin Ser Thr Leu Glu Ser 325 330 335 see*.:Trp Phe Lys Arg *340 2) INFORMATION FOR SEQ ID NO:185: "see:* SEQUENCE CHARACTERISTICS: LENGTH: 326 amino acids TYPE: amino acid 0 STR.ANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein -327- (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Giu Ile Giu Leu 1 5 10 Giu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile 25 Tyr Gln Phe Leu Ser Thr Ile Arg Gin Lys Asp Gly Thr Pro Leu Met Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe Tyr Arg 55 Thr Ilie Asn Leu Met Giu Ala Gly Ilie Lys Pro Val Tyr Val Phe Asp 70 75 Gly Glu Pro Pro Lys Leu Lys Glu Lys Thr Arg Lys Val Arg Arg Giu 90 Met Lys Glu Lys Ala Glu Leu Lys Met Lys Glu Ala Ile Lys Lys Giu 2.00 105 110 Asp Phe Glu Giu Ala Ala Lys Tyr Ala Lys Arg Val Ser Tyr Leu Thr 2.15 2.20 125 Pro Lys Met Val Giu Asn Cys Lys Tyr Leu Leu Ser Leu Met Gly Ile 130 135 140 Pro Tyr Val Glu Ala Pro Ser Glu Gly Giu Ala Gin Ala Ser Tyr Met 145 150 155 160 Ala Lys Lys Gly Asp Val Trp Ala Val Val Ser Gin Asp Tyr Asp Ala 165 170 175 Leu Leu Tyr Gly Ala Pro Arg Val Val Arg Asn Leu Thr Thr Thr Lys ISO 185 190 Giu Met Pro Glu Leu Ile Giu Leu Asn Glu Val Leu Glu Asp Leu Arg 195 200 205 Ile Ser Leu Asp Asp Leu Ile Asp Ile Ala Ile Phe Met Gly Thr Asp 210 215 220 *Tyr Asn Pro Gly Gly Val Lys Gly Ilie Gly Phe Lys Arg Ala Tyr Giu *225 230 235 240 Leu Val Arg Ser Gly Val Ala Lys Asp Val Leu Lys Lys Glu Val Giu 245 250 255 *Tyr Tyr Asp Glu Ile Lys Arg Ile Phe Lys Giu Pro Lys Val Thr Asp *260 265 270 Asn Tyr Ser Leu Ser Leu Lys Leu Pro Asp Lys Glu Gly Ile Ile Lys 275 280 285 *Phe Leu Val Asp Glu Asn Asp Phe Asn Tyr Asp Arg Val Lys Lys His 290 295 300 Val Asp Lys Leu Tyr Asn Leu Ile Ala Asn Lys Thr Lys Gin Lys Thr 305 310 315 320 Leu Asp Ala Trp Phe Lys 325 **328 INFORMATION FOR SEQ ID NO:186: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186: Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Giu Ile Glu Leu Tyr Le u Gly Leu Pro Giu 100 Glu Val1 Glu Gi y Gly 180 Glu Asp Gly Ser Glu 260 Leu Ile Ala Ii Arg Gin Ly Ser His Lei Gly Ile Ly! Lys Lys Gli Lys Trip Arc 105 Tyr Ala Ly! 120 Lys Tyr Lei Glu Gly Gli Ala Val Va' 17( Val Val Arc 185 Leu Aso Gli 200 Asp Ile Ali Gly Ile G1, Lys Asp Va' Ile Phe Ly! 265 Leu Pro Asl 280 Phe Asn Tv: 275 Phe Leu Val Asp Giu Asn 290 329 Val Asp Lys Leu Tyr Asn Leu Ile Ala Asn Tysz TMr T.z I-h -y 3b310 315 320 Leu Asp Ala Trp Phe Lys His His His His His His 325 330 INFORM4ATION FOR SEQ ID NO:1B7: SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187: Met Gly Val Gin Phe Gly Asp Phe Ile Pro Lys Asn Ile Ile Ser Phe 1 5 10 Ala Leu Ala Ile Lys Met 105 Ala Tyr Gly Ser Val1 185 Val Lys Val Lys Gln 265 Ser Lys Asp Pro Leu Ala Lys Phe 260 330 Tyr Ala Ile Lys Giu Phe Phe Leii Th Ast- A. n ayr 275 280 285 Asn Leu Val Trp Arg Asp Pro Asp Giu Giu Gly Ile Leu Lys Phe Leu 290 295 300 Cys Asp Giu His Asp Phe Ser Giu Giu Arg Val Lys Asn Gly Leu Giu 305 310 315 320 Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gin Ser Thr Leu Giu Ser 325 330 335 Trp Phe Lys Arg 340 INFORMIATION FOR SEQ ID NO:i88: SEQUENCE CHARACTERISTICS: LENGTH: 326 amino acids TYPE: amino acid STRANDEDNESS: not reievant TOPOLOGY: not reievant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188: Met Gly Val Pro Ile Gly Gu Ile Ile Pro Arg Lys Glu Ile Giu Leu 1 5 10 Glu Asn Leu Tyr Gly Lys Lys Ile Aia Ile Asp Ala Leu Asn Ala Ile 25 Tyr Gin Phe Leu Ser Thr Ile Arg Gin Lys Asp Giy Thr Pro Leu Met 40 Asp Ser Lys Giy Arg Ile Thr Ser His Leu Ser Giy Leu Phe Tyr Arg 55 Thr Ile Asn Leu Met Giu Ala Gly Ile Lye Pro Val Tyr Val Phe Asp 70 75 Gly Giu Pro Pro Giu Phe Lys Lye Lys Giu Leu Glu Lye Arg Arg Giu 90 Ala Arg Giu Glu Ala Giu Giu Lys Trp Arg Giu Ala Leu Glu Lys Gly *..100 105 110 Giu Ile Giu Giu Ala Arg Lye Tyr Ala Gin Arg Ala Thr Arg Val Asn 115 120 125 Giu Met Leu Ile Giu Asp Ala Lye Lys Leu Leu Giu Leu Met Gly Ile 130 135 140 Pro Ile Val Gin Ala Pro Ser Giu Gly Giu Ala Gin Ala Ser Tyr Met 145 150 155 160 Ala Lye Lye Gly Asp Val Trp Ala Val Val Ser Gin Asp Tyr Asp Ala *165 170 175 Leu Leu Tyr Gly Ala Pro Arg Val Val Arg Asn Leu Thr Thr Thr Lye 180 185 190 **Giu Met Pro Glu Leu Ile Glu Leu Asn Giu Val Leu Giu Asp Leu Arg 195 200 205 Ile Ser Leu Asp Asp Leu Ile Asp Ile Ala Ile Phe Met Gly Thr Asp 210 215 220 331

T

yL: Asn Pro Gly Gly Vali Lys uiy lie Gly Phe Lys Arg Ala Tyr Giu 225 230 235 240 Leu Val Arg Ser Gly Val Ala Lys Asp Val Leu Lys Lys Giu Val Giu 245 250 255 T1yr Tyr Asp Glu Ile Lys Arg Ile Phe Lys Giu Pro Lys Vai Thr Asp 260 265 270 Asn Tyr Ser Leu Ser Leu Lys Leu Pro Asp Lys Giu Gly Ile Ile Lys 275 280 285 Phe Leu Vai Asp Giu Asn Asp Phe Asn Tyr Asp Arg Vai Lys Lys His 290 295 300 Val Asp Lys Leu Tyr Asn Leu Ile Ala Asn Lys Thr Lys Gin Lys Thr 305 310 315 320 Leu Asp Ala Trp Phe Lys 325 332

Claims (31)

1. A mixture comprising: i) a first structure-specific nuclease, wherein said first nuclease comprises a purified archaeal FEN-1 endonuclease, and ii) a purified 5' nuclease.

2. The mixture of claim 1, wherein said first structure-specific nuclease comprises an Archaeoglobusfulgidus FEN-1 endonuclease.

3. The mixture of claim 1, wherein said 5' nuclease comprises an Archaeoglobus fulgidus FEN-1 endonuclease.

4. The mixture of claim 1, wherein said 5' nuclease comprises a polymerase.

The mixture of claim 4, wherein said polymerase comprises a DNA polymerase.

6. The mixture of claim 5, wherein said DNA polymerase comprises a thermostable DNA polymerase.

7. The mixture of claim 1, wherein said 5' nuclease is derived from a DNA polymerase altered in amino acid sequence such that it exhibits reduced DNA synthetic activity from that of the wild-type DNA polymerase but retains substantially the same nuclease activity of the wild-type DNA polymerase.

8. The mixture of claim 7, wherein said 5' nuclease comprises a thermostable nuclease.

9. The mixture of claim 1, wherein said 5' nuclease is selected from the group 2o consisting of CLEAVASE BN enzyme, CLEAVASE DA enzyme, CLEAVASE DN enzyme, CLEAVASE DV enzyme, CLEAVASE BN/thrombin enzyme, CLEAVASE TThDN enzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, Escherichia coli Exo I, and Saccharomyces cerevisiae Radl/RadlO complex. 25

10. A nucleic acid treatment kit comprising: a) a composition comprising purified archaeal FEN-1 endonuclease; b) a first oligonucleotide comprising a 5' portion complementary to a first portion of a target nucleic acid; and Sc) a second oligonucleotide comprising a 5' portion complementary to a second 30 portion of said target nucleic acid downstream of and contiguous to said first portion; and a 3' portion.

11. The kit of claim 10, wherein said 3' portion of said second oligonucleotide comprises a 3' terminal nucleotide not complementary to said target nucleic acid. [R:\LIBI)06779.doc:NSS COMS ID No: SBMI-01013091 Received by IP Australia: Time 12:32 Date 2004-11-25 NOV. 2004 12:26 SPRUSON FERGUSON NO. 9150 P. 334

12. The kit of claim 10, wherein said 3' portion of said second oligonucleotide consists of a single nucleotide not complementary to said target nucleic acid.

13. The kit of claim 10, further comprising a solid support.

14. The kit of claim 13, wherein said first oligonucleotide is attached to said solid suport.

The kit of claim 13, wherein said second oligonucleotide is attached to said solid support.

16. The kit of claim 10, further comprising a buffer solution.

17. The kit of claim 16, wherein said buffer solution comprises a source of 1o divalent cations.

18. The kit of claim 17, wherein said divalent cation comprises Mn t

19. The kit of claim 10, further comprising a third oligonucleotide complementary to a third portion of said target nucleic, acid upstream of said first portion of said first target nucleic acid.

20. The kit of claim 10, further comprising one or more target nucleic acids.

21. The kit of claim 17, wherein said divalent cation comprises Mg~ t

22. The kit of claim 10, wherein said purified FEN-1 endonactease comprises an Archaeoglobusfulgidus FEN- 1 endonuclease.

23. The kit of claim 10, wherein said purified FEN-1 endonuclease comprises a chimerical FEN-l endonuclease.

24. The kit of claim 23, wherein said chimerical FEN-l endonuclease comprises at least a portion of a FEN-i endonuclease selected from the group consisting of Pyrococcus woesei FEN- 1 endonuclease, Pyrococcus-furiosus FEN-i1 endonuclease, Methanococcusj annaschii FEN- 1 endonuclease, Methanobacteriwn thermoautotrophicum FEN- 1 endonuclease, and Archaeoglobusfudgidzss FEN- I endonuclease.

A kit comprising a chimerical FEN-1 endonuclease, wherein said chimerical odes endonuclease comprises at least a portion of an archaeal FEN-lI endonuclease.

26. The kit of claim 25, wherein said chimerical F0N-1 endonuclease comprises at 5 least a portion of a PEN-i endonuclease selected from the group consisting of Pyrococcus 30 woesei PEN-I endonuclease, Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN- 1 cndonuclease, Met hanoba cterium thermoautosrophicum PEN-1 endonuclease, and Archaeoglobusfulgidus FEN- I endonuclease.

27. The kit of claim 25, further comprising: a) a first oligonucleotide, comprising a 5' portion complementary to a first portion of a target nucleic acid; and JRA\LLBFF]06779.doc:NS COMS ID No: SBMI-01 01 3091 Received by IP Australia: Time 12:32 Date 20D4-11-25 NOV. 2004 12:26 SPRUSON FERGUSON NO. 9150 F. 11/11 335 b) a second oligonucleotide comprising a 5' portion complementary to a second portion of said target nucleic acid downstream of and contiguous to said first portion; and a 3' portion.

28. A mixture comprising: i) a first structure-specific nuclease, wherein said first s nuclease comprises a purified archaeal FEN-1 endonuclease; and ii) a purified 5' nuclease, substantially as hereinbefore described with reference to any one of the examples.

29. A nucleic acid treatment kit comprising: a) a composition comprising purified archaeal FEN-I endonuclease; b) a first oligonucleotide comprising a 5' portion complementary to a first portion of to a target nucleic acid, and c) a second oligonucleotide comprising a 5' portion complementary to a second portion of said target nucleic acid downstream of and contiguous to said first portion; and a 3' portion, substantially as hereinbefore described with reference to any one of the examples.

A composition comprising a chimerical FEN-1 endonuclease, wherein said chimerical endonuclease comprises at least a portion of an archaeal FEN-1 endonuclease, substantially as hereinbefore described with reference to any one of the examples.

31. A kit comprising a chimerical FEN-1 endonuclease, wherein said chimerical endonuclease comprises at least a portion of an archaeal FEN-1 endonuclease, substantially as hereinbefore described with reference to any one of the examples. 20 Dated 25 November, 2004 Third Wave Technologies, Inc. 0 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON lI \l.lRFF]06779.doc:NSS I COMS ID No: SBMI-01013091 Received by IP Australia: Time 12:32 Date 2004-11-25