JPH10509322A - Rapid detection and identification of nucleic acid variants and pathogens - Google Patents

Abstract

  (57) [Summary] The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5 'nucleases and 3' exonucleases, are used to screen for known and unknown mutations, such as single base changes in nucleic acids. Methods are provided that allow the identification of genetic mutations in human gene sequences (including the human p53 gene) in a sample. Methods are provided that enable the detection and identification of bacterial and viral pathogens and species in a sample.

Description

DETAILED DESCRIPTION OF THE INVENTION                Rapid detection and identification of nucleic acid variants and pathogens Field of the invention   The present invention provides methods and compositions for treating nucleic acids, particularly nucleic acid sequences and arrangements. Methods and compositions for detecting and characterizing column changes. Background of the Invention   Detection and characterization of specific nucleic acid sequences and sequence changes are indicative of infection. Presence of irs or microbial nucleic acid sequences, mammalian inheritance associated with disease and cancer Detection of the presence of offspring mutants or alleles and found in forensic samples It has been used to identify the origin of nucleic acids, as well as to determine paternity.   The art is concerned with detecting and characterizing specific nucleic acid sequences and sequence changes. There are various known methods that can be used. Nevertheless, the human genome and As the nucleic acid sequence data of genomes of pathogens and pathogens accumulate, it is quick, reliable, and cost-effective. Increased demand for specific sequences that are efficient and user-friendly Continue to do. Importantly, these tests will produce very low copies of the sequence of interest. Must be able to generate a detectable signal from the . The following discussion discusses the three stages of nucleic acid detection currently in use, namely, I.I. Rare distribution Signal amplification techniques for detection of columns, II. For detection of higher copy number sequences Direct detection technology, and III. Sequence change somewhere within a defined DNA fragment Detection of unknown sequence changes for rapid screening of DNA.   I. Signal amplification technology method for amplification   "Polymerase chain reaction" (PCR) involves a first generation method of nucleic acid amplification. However While using the same specificity principle but generating signals with different amplification mechanisms Some other methods have been developed. These methods include "ligase chain reaction. Response (LCR), Self-Sustained Synthetic Reac tion) ”(3SR / NASBA) and“ Qβ-Replicase ”(Qβ) .   Polymerase chain reaction (PCR)   The polymerase chain reaction (PCR) is described in Mullis and Mullis et al., U.S. Pat. Cloning or purification as described in 195 and 4,683,202 Methods to increase the concentration of segments of a target sequence in a genomic DNA mixture U. This technique offers one approach to the problem of low target sequence concentration . PCR is used to directly increase the target concentration to a level that is easily detectable. Can be used. This method of amplifying a target sequence contains the desired target sequence. The resulting DNA mixture contains a 1 molar excess of 2 complementary to each strand of the double-stranded target sequence. And introducing two oligonucleotide primers. Denaturing the mixture And then hybridized. After hybridization, the primer is It is extended with merase to form a complementary strand. Relatively high concentrations of the desired target sequence Denaturation, hybridization, and polymerase extension The process can be repeated as many times as necessary.   The length of the segment of the desired target sequence is relative to each of the primers. Can be determined by the position, so this length is a controllable parameter It is. The sequence in which the desired segment of the target sequence predominates in the mixture (in terms of concentration) They are said to be "PCR amplified".   Ligase chain reaction (LCR or LAR)   Ligase chain reaction (LCR: Barany, Proc. Natl. Acad. Sci., 88: 189 (1991);  Barany, PCR Methods and Applic., 1: 5 (1991) and Wu and Wallace, Genomi. cs 4: 560 (1989) described as "ligase amplification reaction" (LAR) Has evolved into another well-recognized method of nucleic acid amplification. LCR Four oligonucleotides (ie, only one strand of the target DNA Hybridize with two adjacent oligonucleotides that form a bridge and the opposite strand (A complementary set of adjacent oligonucleotides to form) and mix DNA ligase Add to the mixture. If there is complete complementarity at the junction, the ligase will hybridize. Each set of code forming molecules will be covalently linked. The important thing is that in LCR Probes base-pair with sequences in target sample without gaps or mismatches Only if they are connected to each other. Denaturation, hybridization and And ligation cycles are repeated to amplify short DNA segments . LCR can also be combined with PCR to enhance detection of single base changes. Use (Segev, PCT International Publication WO09001069 A1 (1990)). However, The four oligonucleotides used in this assay are paired into two short ligations. Generates a target-independent background signal as it can form possible fragments there is a possibility. The use of LCR for mutant screening involves the use of specific nucleic acids Limited to location surveys.   Self-sustained synthesis reaction (3SR / NASBA)   Self-sustained sequence replication reaction (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87: 1874-1878 [1990], and Proc. Natl. Acad. Sci., 87: 7797 [1990]. The errata shows that RNA sequences can be amplified exponentially at a certain temperature. A transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86: 1173-1). 177 [1989]). The amplified RNA can then be used for mutation detection (Fahy et al., P. CR Meth. Appl., 1: 25-33 [1991]). In this method, the 5 'end of the sequence of interest is Oligonucleotides to add a large RNA polymerase promoter Use primers. Second primer, reverse transcriptase, ribonuclease H, R Contains NA polymerase and ribo- and deoxyribonucleoside triphosphates Iterative circulation of transcription, cDNA synthesis and second strand synthesis in a reaction mixture of enzyme and substrate To amplify the region of interest. For detecting mutations The use of 3SR is useful for screening small DNA segments (eg, 200-300 base pairs). Kinetic.   Q-beta (Qβ) replicase   In this method, a probe recognizing a sequence of interest is used for Qβ replicase. To a replicable RNA template. Of non-hybridized probe A major identified problem with false positives arising from replication is sequence-specific linkage. Presented through the use of a knotting process. However, available thermostable DNA Ligase is not effective on this RNA substrate, so the ligation is It must be performed at low temperature (37 ° C.) with ligase. Because of this, gain specificity The use of elevated temperatures as a means to prevent It can be used to detect mutations in the binding site only (and nowhere else).   Table 1 below lists some of the desirable features for systems useful in sensitive nucleic acid diagnostics. And summarize the capabilities of each of the major amplification methods (Landgren, Trends in Gen etics 9: 199 [1993]).   For a diagnostic method to be successful, it must be very specific. A direct way to control the specificity of nucleic acid hybridization controls the temperature of the reaction It is because of that. The 3SR / NASBA and Qβ systems are all large signal At least one of the enzymes involved in each Cannot be used at high temperatures (ie above 55 ° C.). Therefore, the non-specificity of the probe The reaction temperature cannot be increased to prevent dynamic hybridization. Plow Shortening the probe to make it easier to melt Is more likely to be present in the composite genome. For these reasons, PCR And LCR dominates the field of detection technology research.   The basis for amplification methods in PCR and LCR is that one cycle of product is A template that can be used in all subsequent cycles, and as a result, That is, the number is doubled. The final yield of any such double system is ( 1 + X)ⁿ= Y [where “X” is the average efficiency (the ratio of replicated in each cycle) and “n "Indicates the number of cycles, and" y "indicates the total efficiency or the reaction yield.] (Mullis, PCR Methods Applic., 1: 1 [1991]). Each copy of the target DNA is If used as a template in each cycle of the merase chain reaction, the average efficiency is 100 %. After 20 cycles of PCR, the yield is 2 starting materials.²⁰I.e. 1, 048,576 copies. Do not reduce the average efficiency to 85% by the reaction conditions. Showed that the 20 cycle yield was only 1.85 of the starting material.²⁰Ie, 220,5 13 copies. That is, PCR performed at 85% efficiency is 100% efficient. Only 21% of the final product is obtained compared to the reaction performed. Average efficiency is 5 Reactions reduced to 0% give less than 1% of possible product.   In practice, the normal polymerase chain reaction rarely gives the theoretical maximum yield However, PCR is usually performed for 20 cycles or more to compensate for the low yield. Theoretically 20 To obtain a million-fold amplification obtained in cycles, 34 cycles at 50% average efficiency. At lower efficiencies, the number of cycles required is extraordinary. Ma Also, if the background product is amplified with better average efficiency than the desired target, Any background products will be the predominant products.   In addition, the length and secondary structure of the target DNA, the length and design of the primer, The concentration of immersion and dNTPs, buffer composition, etc. A number of variables can affect the average efficiency of the PCR. Exogenous DNA (eg, laboratory Consideration should also be given to contamination or cross-contamination of the reaction by DNA spilled on the surface. It is a point. Reaction conditions are for each different primer pair and target sequence It must be carefully optimized, and even experienced researchers can May also spend. Difficulties in this method involving numerous technical considerations and other factors Due to gender, a significant obstacle appears in the use of PCR in clinical settings. In fact, still PC R has not penetrated the clinical market in a significant way. Different oligonucleotides for each target sequence Since the LCR must also be optimized to use the leotide sequence, the LCR But the same concerns arise. In addition, both methods provide high temperature Requires expensive equipment.   Many applications of nucleic acid detection technology (e.g., when studying allelic variants) Nucleotide differences as well as detection of specific sequences in Also includes discrimination between sequences that have little or no single nucleotide difference. For PCR A method for detecting a more allele-specific mutation is to use the template strand and the 3 ′ end of the primer. It is difficult for Taq polymerase to synthesize DNA strand when there is a mismatch between Based on the fact that Allele-specific mutation is one of the possible alleles It can also be detected by using a primer that perfectly matches with only. I mean , A mismatch with the other allele acts to inhibit extension of the primer Thereby inhibiting amplification of the sequence. The base composition of the mispairing is Affects the ability to inhibit elongation beyond, and some mismatches inhibit elongation. This method has inherent limitations in that it has no or minimal impact (Kwok Et al., Nucl. Acids Res., 18: 999 [1990]).   A similar 3 'mismatch strategy can be used more effectively to prevent ligation in LCR. (Barany, PCR Meth. Applic., 1: 5 [1991]). In any case, the heat resistant rig Effectively inhibits the action of Lase, but still LCR is a target-independent background It has the disadvantage that amplification starts with the round ligation product. Also, for each position Combining LCR followed by PCR to identify nucleotides is also an option. Clearly a tricky proposal to the floor lab.   II. Direct detection technology   If a sufficient amount of nucleic acid is available to be detected, more copies of that target Direct detection of the sequence without creating a sequence (e.g., as in PCR and LCR). There is an advantage that you can get out. Most notably, the signal is exponentially amplified. The method that is not used is easier to adapt to quantitative analysis. Even a single oligonucleotide Even if multiple dyes are attached to the otide to enhance the signal, The correlation between degree and target amount is straightforward. Such a system is a reaction product that The product itself does not further accelerate the reaction, and contamination of the laboratory surface with the product is a serious problem. It has the additional advantage of not having to. Northern blotting, Southern block In traditional methods of direct detection, such as ligating and ribonuclease protection assays, Usually, radioactivity must be used, and it is not easy to automate. Recently devised The technology avoids the use of radioactivity and / or increases sensitivity in a form that can be automated. I'm trying to get it up. For example, "Cycling Probe R" eaction) "(CPR) and" Branched DNA "(bDNA). It is.   The cycling probe reaction (CPR) (Duck et al., BioTech., 9: 142 [1990]) A long chimeric oligonucleotide consisting of RNA at the center and DNA at both ends use. The probe is hybridized with the target DNA, and Exposure to ase H digests the RNA portion. Because of this, the remaining two The heavy helical DNA is destabilized and the remaining probe is released from the target DNA. , The process is repeated with another probe molecule. Cleavage probe molecular form The signal accumulates at a linear rate. The signal is increased by the repeated steps, The RNA portion of the oligonucleotide is a ribonucleic acid that can be carried by sample preparation. Vulnerable to Aase attacks.   The branched DNA (bDNA) described in Urdea et al., Gene 61: 253-264 (1987) is: Branching structures that carry 35-40 labels on each individual oligonucleotide (Eg, alkaline phosphatase enzyme). this Increases the signal from the hybridization event, but increases the signal from non-specific binding. The signal increases as well.   III. Detect unknown sequence changes   The need for tests that can detect specific nucleic acid sequences and sequence changes It is increasing rapidly with diagnosis. Nucleic acid sequences for genes from humans and pathogens As column data accumulates, rapid and costly unknown mutations in specific sequences The demand for highly efficient and easy-to-use tests is rapidly increasing.   A few ways to scan nucleic acid segments for mutations I have. One option is to determine the entire gene sequence of each test sample (eg, bacterial isolate) It is to be. If the sequence is less than about 600 nucleotides, the amplified material This may be done by using (eg, a PCR reaction product). This And eliminates the time and expense associated with cloning the segment of interest. it can. However, special equipment and highly trained personnel are required, The law is too labor-intensive and uneconomic, making it impractical and effective in clinical settings not.   Given the difficulties associated with sequencing, the number of nucleic acid segments Or at other stages. At the lowest resolution, the same The size of the molecule can be determined by comparison with a known standard run on the same gel . Prior to electrophoresis, multiple restriction enzymes can be constructed so that an ordered map can be constructed. By cleavage in combination, a more detailed molecular image can be obtained. The cut The presence of specific sequences within the strip can be detected by hybridization of the labeled probe. Or in the presence of partial chemical degradation or chain terminating nucleotide analogs The exact nucleotide sequence can be determined by primer extension at   In the case of detection of single base differences between the same sequences, the highest resolution Often needed. The location of the nucleotide in question is known in advance If so, how do you look for single base changes without direct sequencing? That method has been developed. For example, if the mutation of interest happens to be a restriction recognition If located in the column, changes in digestion pattern can be used as diagnostic means ( Example, restriction fragment length polymorphism [RFLP] analysis).   Single point mutations have also been detected by the creation or disruption of RFLP. Mismatch site Mutation was detected by the presence and size of the RNA fragment generated by cleavage at Is determined. Single Nucleotide in DNA Heteroduplex Due to Several Chemicals Peptide mismatches are also recognized and cleaved, resulting in `` Mismatch Chlo emical Cleavage) "(MCC) (Gogos et al., Nucl. Acids Res., 18: 6807-6817 [1990]. ) Provides another strategy for detecting single base substitutions, commonly referred to as It is. However, this method is very toxic, not suitable for use in clinical laboratories Two chemicals, osmium tetroxide and piperidine, must be used. Must.   RFLP analysis is insensitive and requires large samples. For point mutation detection When using RFLP analysis, by its nature, it is known to be a restriction endonuclease. It is limited to the detection of single base changes located within the restriction sequences of ase. In addition, The majority of available enzymes have 4-6 base pair recognition sequences and are frequently cleaved too often Not suitable for large-scale DNA manipulation (Eckstein and Lilley eds., Nucleic Acids and  Molecular Biology, vol. 2, Springer-Verlag, Heidelberg [1988]). Therefore , Since most mutations are not located at such sites, Combination Applicable only to   A small number of rare cleavage restriction enzymes having eight base pair specificities have been isolated, Although they are widely used in genetic mapping, these enzymes are in very small numbers, Limited to recognition of G + C-rich sequences, cleavage at sites that are highly prone to cluster formation (Barlow and Lehrach, Trends Genet., 3: 167 [1987]). Recently, 12 or more Encoded by a group I intron thought to have a base pair specificity of Endonuclease was discovered (Perlman and Butow, Science 246: 1106 [19 89]), again, these are very few.   If the change is not within the recognition sequence, a primer extension or ligation event is paired or High near an unknown nucleotide so that it can be used as an indicator of mismatch Designing allele-specific oligonucleotides (ASO) to form a bridge Can be In addition, radiolabeled allele-specific oligonucleotides (AS O) has been applied to the detection of specific point mutations (Co nner et al., Proc. Natl. Acad. Sci., 80: 278-282 [1983]). This method uses a single Based on differences in melting temperatures of short DNA fragments differing in leotide. Astringent hybrid Pad and wash conditions distinguish between mutant and wild-type alleles . Also, the ASO approach applied to the PCR product was a ras gene and gsp / By various investigators to detect and characterize point mutations in the gip oncogene It is widely used (for the ras gene, see Vogelstein et al., N. Eng. J. Med., 31). 9: 525-532 [1988] and Farr et al., Proc. Natl. Acad. Sci., 85: 1629-1633 [1988] For the gsp / gip oncogene, see Lyons et al., Science 249: 655-659 [1990]). Various Multiple possible oncogene mutations due to multiple nucleotide changes In order to cover the differences, the ASO method uses a large number of oligonucleotides. Need to be   The use of any of the above techniques (ie, RFLP and ASO) is The exact location of the mutation must be known prior to testing. That is, They are suddenly unknown in their nature and location within the gene or sequence of interest. However, it cannot be applied when it is necessary to detect the presence of the mutation.   The other two methods detect changes in electrophoretic mobility in response to small sequence changes It is because of that. `` Denaturing Gradient Gel Ele ctrophoresis) (DGGE), one of these methods is electrophoresis on gradient gels. Slightly different sequences show different patterns of local melting when separated by electrophoresis It is based on the observation that Thus, different nucleotides at a single nucleotide Differences in the melting properties of mono- and heteroduplexes may lead to the presence of mutations in the target sequence. Mutants can be identified because they can be detected by corresponding changes in their electrophoretic mobilities. You can do it. Complete denaturation of the sequence of interest without complete strand dissociation As described above, the fragment to be analyzed (usually the PCR product) is composed of long extending GC bases. It is "clamped" at one end by pairs (30-80). The GC “clamp” is recognized by DGGE by binding to the DNA fragment. The rate of possible mutations increases (Abrams et al., Genomics 7: 463-475 [1990]). Increase To ensure that the width array has a low dissociation temperature, one GC clamp It is important to attach to the primers (Sheffield et al., Proc. Natl. Acad. Sc. i., 86: 232-236 [1989], and Lerman and Silverstein, Meth. Enzymol., 155 : 482-501 [1987]). Modification of the technique using temperature gradients has also been developed (Wa rtell et al., Nucl. Acids Res., 18: 2699-2701 [1990]). It can also be applied to main strands (Smith et al., Genomics 3: 217-223 [1988]).   One limitation of the utility of DGGE is that denaturing conditions must be met for each type of DNA tested. There is a need to optimize. Further, the method further comprises: Special equipment is required to prepare and maintain the required high temperatures during electrophoresis. The clamping tail (c) on one oligonucleotide for each sequence tested The costs associated with synthesizing the lamping tail are also a major consideration. Also , DGGE requires a long operation time. The long operating time of DGGE is Called constant denaturant gel electrophoresis (CDGE) Truncation in the modification of DGGE (Borrensen et al., Proc. Natl. Acad. Sci. USA 88: 8405 [1991]). High in CDGE due to detection of unknown mutations To obtain efficiency, it is necessary to use the gel under different denaturant conditions.   Temperature gradient gel electrophoresis (TGG In a technique similar to DGGE called E), a temperature gradient rather than a chemical denaturant gradient is used. (Scholz et al., Hum. Mol. Genet., 2: 2155 [1993]). In TGGE, the electric field Special equipment must be used to create a vertically oriented temperature gradient No. TGGE can detect mutations in relatively small DNA fragments Therefore, scanning large gene segments requires multiple PCR products before using the gel. It is necessary to use a product.   "Single-Strand Conformation Polymorphism ) "(SSCP) is another common method described by Hayashi, Sekya et al. (Hayash PCR Meth. Appl., 1: 34-38 [1991]) Allows a single-stranded nucleic acid to adopt a characteristic conformation in non-denaturing conditions. Based on the observation that these conformations affect electrophoretic mobility. It is a spider. The complementary strands are sufficiently different that one strand is separated from the other. Take a structure that: Alterations in the sequence within the fragment also alter the conformation. As a result, it alters the mobility, which can be used as an assay for sequence variation. (Orita et al., Genomics 5: 874-879 [1989]).   The SSCP method uses a DNA segment labeled on both strands (eg, a PCR product). Denaturation followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel. Release (allows intramolecular interactions to form, which are disturbed during operation) Not to include). This technique is insensitive to changes in gel composition and temperature. Always sensitive. An important limitation of this method is that it combines data from different laboratories. It is relatively difficult to compare under apparently similar conditions.   Dideoxy fingerprinting (ddF) has been described for the presence of unknown mutations. Another technique developed for scanning genes (Liu and Sommer , PCR Methods Appli., 4:97 [1994]). In the ddF technology, the Sanger dideoxy distribution The components of the column determination method are combined with SSCP. One dideoxy termine The dideoxy sequencing reaction was carried out using a Similarly, the reaction product is subjected to electrophoresis on a non-denaturing polyacrylamide gel, A change in the mobility of the connection segment is detected. ddF is SSC at the point where sensitivity is increased. P is an improvement over P, but ddF requires the use of expensive dideoxynucleotides. However, this technique uses fragments of a size suitable for SSCP (ie, optimal detection of mutations). (200-300 base fragment) for analysis.   In addition to the foregoing limitations, all of these methods relate to the size of Limited. The direct sequencing approach requires that all fragments be included in the coverage. , Cloning is required for sequences longer than 600 base pairs, resulting in It involves delays and expense for cloning or primer walking. S SCP and DGGE are subject to even more severe size restrictions. Against sequence changes These methods are not considered appropriate for larger fragments due to reduced sensitivity Can be SSCP reports that it detects 90% of single base substitutions within a 200 base pair fragment As reported, the detection dropped to less than 50% for the 400 bp fragment. U. Similarly, the sensitivity of DGGE decreases as fragment length reaches 500 base pairs. Less. Also, the ddF technique is used in the case of a combination of direct sequencing and SSCP. Similarly, the DNA is limited to DNA of a relatively small size that can be screened.   Obviously, the size is so sensitive that all genes can be analyzed, not gene fragments. There is still a need for an insensitive method. It also has a variety of backgrounds and technologies Such a means is available so that researchers can compare data obtained from different laboratories. Must be robust. Ideally, such a method would be "multiplexing" (I.e., several molecules or genes performed in a single reaction or gel lane Simultaneous analysis, usually separated from each other by identifying labels or probes ) Would be compatible. Such analytical techniques will be Facilitates the use of internal standards for data comparison and increases personnel and equipment productivity I will let you. This ideal method would also be easy to automate.   Summary of the Invention   The present invention relates to methods and compositions for treating nucleic acids, particularly in human gene sequences. And characterize nucleic acid sequences and sequence changes in genetic and microbial gene sequences Methods and compositions. The present invention relates to a nucleic acid cleavage structure in a site-specific manner. Providing a means for cleaving. In one embodiment, the cleavage means comprises a nucleic acid group Is an enzyme that has the ability to cleave cleavage structures on proteins, Form the basis of the delivery method. The invention relates, inter alia, to 1) mutations in human gene sequences. And 2) detection and identification of pathogenic organisms, including but not limited to I) The use of the novel detection method for clinical diagnostic purposes is contemplated.   In one embodiment, the invention relates to native (ie, "wild-type") DNA polymerases. The sequence has been modified in comparison to the native sequence to show altered DNA synthesis activity from Encoding a modified DNA polymerase (ie, a "mutant" DNA polymerase) Intended DNA sequence. For the polymerase, no synthesis should take place. Is not necessary, but it inhibits the method in the absence of polymerase activity It is desired that a cleavage reaction occurs at the level. The encoded DNA polymerase Is modified to show reduced synthetic activity from the natural DNA polymerase. Preferably. Thus, the enzyme of the present invention is a nuclease, It has the ability to specifically cleave nucleic acids. Importantly, the nucleases of the present invention The ability to cleave the cleaved structure to produce a separate cleavage product.   The present invention contemplates nucleases from various sources, including thermostable nucleases. Is done. Thermostable nucleases cause nucleic acid hybridization to occur very specifically Operable at temperature, enabling allele-specific detection (including single base mismatches) To be particularly useful. In one embodiment, the thermostable 5 'nucleus is These are Thermus aquaticus, Thermus flavus (Th ermus flavus) and Thermus thermophilus ( (Non-limiting) Natural polymerers of various Thermus species Selected from the group consisting of modified polymerases derived from zeolites.   The present invention is not limited to the use of thermostable nucleases. In this specification As shown, in the method of the present invention, nucleases from mesophilic organisms (eg, E. coli (E. coli) i) Exo III, Saccharomyces cerevisiae Rad1 / Rad1 0 complex).   The present invention relates to methods for detecting and characterizing nucleic acid sequences and sequence changes. Use creatase. The present invention cleaves nucleic acid cleavage structures in a site-specific manner Means for. Scan for known and unknown mutations, such as single base changes in nucleic acids. Nuclease activity is used for cleaning.   In one embodiment, the present invention relates to a method of treating nucleic acids, comprising: a) i) a cleavage procedure. And ii) providing a nucleic acid substrate, b) said nucleic acid substrate producing one or more cleavage structures. Treating said substrate under conditions such as: and c) reacting said cleavage means with said cleavage structure. To obtain one or more cleavage products.   In one embodiment, the cleavage means is an enzyme. In a preferred embodiment, the open The cleavage means is a nuclease. In another preferred embodiment, the nucleus Ze is a Cleavase^TMBN enzyme, Thermus aquaticus quaticus) DNA polymerase, Thermus thermophils us) DNA polymerase, Escherichia coli Exo III and Saccharomyces Selected from the group consisting of the Saccharomyces cerevisiae Rad1 / Rad10 complex. Devour.   The nucleic acid substrates include 7-deaza-dATP, 7-deaza-dGTP and dUTP. Nucleotide analogs selected from the group consisting of, but not limited to, It is intended to include In one embodiment, the nucleic acid of the step is essentially single-stranded . It is not intended that the nucleic acid substrate be limited to any particular form; The acid substrate is intended to be single-stranded or double-stranded DNA or RNA.   In one embodiment, when using a double-stranded nucleic acid substrate, the processing step (b) is Making the single-stranded nucleic acid essentially single-stranded, and wherein the single-stranded nucleic acid has a secondary structure or characteristic Exposing the single-stranded nucleic acid to conditions that result in a highly folded structure. In one preferred embodiment, the temperature is increased to essentially eliminate the double-stranded nucleic acid. Single-stranded.   In another embodiment, the method comprises detecting one or more cleavage products as described above. Further comprising the step of:   In a preferred embodiment, the nucleic acid substrate is an oligonucleotide containing a human p53 gene sequence. Gogonucleotides. In another embodiment, the nucleic acid substrate is a microbial gene. Includes oligonucleotide containing sequence.   The present invention further relates to a method for treating nucleic acids, comprising: a) a) a solution comprising manganese. C) providing a nucleic acid substrate, and b) treating the nucleic acid substrate at an elevated temperature. C) reducing the temperature under conditions such that the substrate forms one or more cleavage structures. D) reacting the cleavage means with the cleavage structure to obtain one or more cleavage products; e) detecting the cleavage product. In this case too Was The cleavage means may be an enzyme. As mentioned above, the cleavage means is a nuclease. There may be. In another preferred embodiment, the nuclease is cleaved. Alease^TMBN enzyme, Thermus aquaticus DN A polymerase, Thermus thermophilus DNA polymerase Merase, Escherichia coli Exo III and Saccharomyces cerevisiae D (Saccharomyces cerevisiae) selected from the group consisting of Rad1 / Rad10 complex.   The nucleic acid substrates include 7-deaza-dATP, 7-deaza-dGTP and dUTP. Nucleotide analogs selected from the group consisting of, but not limited to, It is intended to include In one embodiment, the nucleic acid of the step is essentially single-stranded . It is not intended that the nucleic acid substrate be limited to any particular form; The acid substrate is intended to be single-stranded or double-stranded DNA or RNA.   In a preferred embodiment, the nucleic acid substrate is an oligonucleotide containing a human p53 gene sequence. Gogonucleotides. In another embodiment, the nucleic acid substrate is a microbial gene. Includes oligonucleotide containing sequence.   The present invention further provides a method for detecting a mutation in the human p53 gene, a) providing a cleavage means, and ii) a nucleic acid substrate comprising a human p53 gene sequence, b) Treating the substrate under conditions such that the nucleic acid substrate produces one or more cleavage structures; c. C) reacting said cleavage means with said cleavage structure to obtain one or more cleavage products; Comparing the cleavage product with the cleavage product obtained by cleavage of a reference p53 gene sequence A method comprising:   In a preferred embodiment, the cleavage generated by cleavage of the reference p53 gene sequence Are selected from the group consisting of SEQ ID NOs: 79-81, 84-89 and 94-97 Generated by cleavage of a nucleic acid substrate containing the human p53 gene sequence. In this specification Provides an additional p53 mutant sequence, wherein SEQ ID NO: 79 contains the wild-type p53 cDNA Describe the sequence. Table 2 below shows the identity and position of a number of known p53 mutations. Position. Combining the information in Table 2 with the sequence of the wild-type p53 cDNA of SEQ ID NO: 79 As a result, cDNAs corresponding to a number of p53 mutations listed in Table 2 were obtained. Complete nucleotide sequences can be obtained. In addition, it is fully explained in this specification. As will be appreciated, the method of the present invention is not yet limited to human gene sequences such as the human p53 gene. Special Enables screening or "scanning" for unmarked mutations .   The present invention also relates to one or more alleles of the human p53 gene. A method for producing a reference library of gene fingerprints (for diagnostics) And a) i) a cleavage means, and ii) a nucleic acid substrate derived from a human p53 gene sequence. , B) determining whether the nucleic acid substrate and the cleavage means form one or more secondary structures with the extracted nucleic acid. Under conditions such that the cleavage means cleaves the secondary structure to produce a plurality of cleavage products. C) separating said plurality of cleavage products, and d) separating said separated products. Maintaining a recordable testable standard for cleavage products. You.   The term "gene fingerprint" refers to changes in the nucleic acid sequence (e.g., deletions, insertions, Or single point substitution) alters the formation structure and thereby the band pattern (i.e. `` Inger print '' or `` barcode '') to express the sequence differences. And allowing rapid detection and identification of the mutant.   The invention also relates to one or more pairs of one or more genes from a eukaryote (eg, a mammal). Generic fingerprint recording criteria characteristic of progeny (i.e., diagnostic) A method for producing an library, comprising: a) i) a cleavage means; and ii) a eukaryotic gene. B) providing a nucleic acid substrate derived from one or more alleles of The extracted nucleic acid forms one or more secondary structures and the cleavage means comprises Are cleaved to produce a plurality of cleavage products, and c) And d) a testable description of said separated cleavage product Recording standards are intended.   The present invention also provides a method for identifying a strain of a microorganism, comprising: a) i) a cleavage means; And ii) providing a nucleic acid substrate comprising a sequence derived from one or more microorganisms, b) the nucleic acid substrate Treating the substrate under conditions such that produces one or more cleavage structures; and c) Reacting a cleavage means with the cleavage structure to obtain one or more cleavage products. Intended way.   Preferred cleavage means are enzymes such as nucleases. Successful method of the present invention Examples of enzymes that can be used for, for example, Cleavase^TMBN enzyme , Thermus aquaticus DNA polymerase, Thermus ・ Thermus thermophilus DNA polymerase, Escherichia coli (Escheri ch ia coli) Exo III, Saccharomyces cerevisiae Rad Examples include, but are not limited to, the 1 / Rad10 complex.   The nucleic acid substrates include 7-deaza-dATP, 7-deaza-dGTP and dUTP. Nucleotide analogs selected from the group consisting of, but not limited to, It is intended to include In one embodiment, the nucleic acid of the step is essentially single-stranded . It is not intended that the nucleic acid substrate be limited to any particular form; The acid substrate is intended to be single-stranded or double-stranded DNA or RNA.   In one embodiment, when using a double-stranded nucleic acid substrate, the processing step (b) is Making the single-stranded nucleic acid essentially single-stranded, and wherein the single-stranded nucleic acid has a secondary structure or characteristic Exposing the single-stranded nucleic acid to conditions that result in a highly folded structure. In one preferred embodiment, the temperature is increased to essentially eliminate the double-stranded nucleic acid. Single-stranded.   In another embodiment, the method comprises detecting one or more cleavage products as described above. Further comprising the step of:   The microorganism of the present invention is intended to be selected from various microorganisms. The present invention It is not intended to be limited to that particular type of microorganism. Conversely, the present invention relates to bacteria, Microorganisms, including (but not limited to) fungi, protozoa, ciliates and viruses It is intended to be performed using living organisms. The microorganism is of a specific genus, species, strain or serum It is not intended to be limited to types. In fact, the bacteria are Campylobacter bacter), Escherichia, Mycobacterium ), Salmonella, Shigella and Staphylococca Group consisting of (but not limited to) members of the genus Staphylococcus It is intended to be chosen from. In one preferred embodiment, the microorganism is a multidrug Includes strains of resistant Mycobacterium tuberculosis. Also, the present invention Including but not limited to hepatitis C virus and simian immunodeficiency virus (Not) using a virus.   Another embodiment of the present invention is a method for detecting and identifying a strain of a microorganism. Extracting nucleic acids from a sample suspected of containing one or more microorganisms; The extracted nucleic acid forms one or more secondary structures and the cleavage means Contacting under conditions such that the means cleaves the secondary structure to produce one or more cleavage products. A method comprising the step of:   In one embodiment, the method further comprises the step of separating the cleavage product. No. In yet another embodiment, the method comprises the steps of detecting the cleavage product. Step.   In one preferred embodiment, the present invention provides a method for isolating said extract isolated from said sample. The detected cleavage product obtained from the cleavage of the output nucleic acid is derived from one or more reference microorganisms. Further comprising comparing the isolated cleavage products obtained from the cleavage of the nucleic acid. So In such a case, the sequence of the nucleic acid from one or more reference microorganisms may be related Different (eg, mutated sequence or suddenly known or already characterized) Wild-type control for the mutant sequence).   In another preferred embodiment, the present invention provides the method of the invention, wherein To obtain a nucleic acid substrate, and contacting the substrate with the cleavage means Further comprising a step. In one embodiment, isolating the polymorphic locus comprises nucleic acid amplification. Perform by width. The invention is limited by the method of nucleic acid amplification used. Nucleic acid amplification One way to do this is the polymerase chain reaction. In another embodiment, 7- The group consisting of deaza-dATP, 7-deaza-dGTP and dUTP (to these The nucleic acid in the presence of a nucleotide analogue selected from, but not limited to) Do width. The nucleic acid amplification (e.g., PCR) comprises: 1) a common gene from the polymorphic locus An oligonucleotide primer that matches the sequence (i.e., the primer (Including the same sequence found on the strand of nucleic acid from the locus), or 2) the polymorphism Oligonucleotide primers complementary to the consensus gene sequence from the That is, they are the complement of a strand of nucleic acid from the polymorphic locus). Is intended. In one embodiment, the polymorphic locus is a ribosomal RNA gene. Including children. In a particularly preferred embodiment, the ribosomal RNA gene is a 16S ribosome. It is a somatic RNA gene.   In one embodiment of the method, the cleavage means is an enzyme such as a nuclease. . In a particularly preferred embodiment, the nuclease is Cleavase.^TM BN, Thermus aquaticus DNA polymerase, Term S ・ Thermus thermophilus DNA polymerase, Escherichia coli (Escheri chia coli) Exo III and Saccharomyces cerevisi ae) selected from the group consisting of (but not limited to) the Rad1 / Rad10 complex You. In addition, the enzyme is a euthermic bacterium [this is Thermus aquaticus (Thermus aquaticus), Thermus flavus and Thermus thermophile Selected from the group consisting of Rus (Thermus thermophilus)] Has a portion of the amino acid sequence that is homologous to a portion of the amino acid sequence of merase It is also intended to be good.   The nucleic acid substrate is not intended to be limited to any particular form; The acid substrate is intended to be single- or double-stranded RNA or DNA. Double strand When using a nucleic acid substrate, the processing step of the method essentially converts the double-stranded nucleic acid to single-stranded. Subjecting the single-stranded nucleic acid to conditions such that the single-stranded nucleic acid assumes a secondary structure. May be included. In one preferred embodiment, the temperature is increased This makes the double-stranded nucleic acid essentially single-stranded.   The microorganism of the present invention is intended to be selected from various microorganisms. The present invention It is not intended to be limited to that particular type of microorganism. Conversely, the present invention relates to bacteria, Microorganisms, including (but not limited to) fungi, protozoa, ciliates and viruses It is intended to be performed using living organisms. The microorganism is of a specific genus, species, strain or serum It is not intended to be limited to types. In fact, the bacteria are Campylobacter bacter), Escherichia, Mycobacterium ), Salmonella, Shigella and Staphylococca Group consisting of (but not limited to) members of the genus Staphylococcus It is intended to be chosen from. In one preferred embodiment, the microorganism is multi-drug resistant Of Mycobacterium tuberculosis. In addition, the present invention Influenza C virus and simian immunodeficiency virus (including but not limited to Is not intended to be performed using a virus.   Also, the present invention provides a method for characterizing (ie, identifying) one or more alleles of various microorganisms. A method for producing a reference library of gene fingerprints, Providing a nucleic acid substrate derived from a microbial gene sequence with the cleavage means and the nucleic acid substrate and the cleavage means The extracted nucleic acid forms one or more secondary structures and the cleavage means comprises Are contacted under conditions such that the cleavage results in a plurality of cleavage products, The cleaved product is separated and a testable record basis for the separated cleaved product is established. A process comprising the step of maintaining is contemplated.   The present invention is not intended to be limited by the nature of the microorganism. The detection and Identification is applicable to all microorganisms, including viruses and bacteria.   In addition, the present invention also relates to gene fingers characteristic (i.e., for diagnosis) of pathogenic microorganisms. A method for producing print recording standards (eg, libraries), comprising: a) i) a cleavage means; And ii) a characteristic (e.g., from a polymorphic locus) nucleus isolated from a known pathogenic microorganism Preparing an acid substrate, b) combining said nucleic acid substrate and cleavage means with said extracted nucleic acid in one or more secondary structures. Forming a structure and the cleavage means cleaving the secondary structure to produce a plurality of cleavage products. C) separating said plurality of cleavage products, and d) Maintaining testable record standards for the separated cleavage products of the above. Intended manufacturing method.   The present invention also provides a kit for treating a nucleic acid, comprising: a) reacting with a cleavage structure to produce a cleavage product; A kit comprising a product-forming enzyme and b) a solution containing manganese. Figure. The enzyme of the kit may be a nuclease. In a preferred embodiment , The nuclease is a Cleavase^TMBN enzyme, Termus aquachi Cousin (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Ther mus thermophilus) DNA polymerase, Escherichia coli Exo III and And Saccharomyces cerevisiae Rad1 / Rad10 complex Selected from the group consisting of (but not limited to): The invention relates to nucleic acid processing Other reagents useful for are contemplated. For example, the kit detects the cleavage product. May be included. The kit further includes a salt solution (eg, KCl and And NaCl solution), manganese chloride solution, buffer solution, and terminate the cleavage reaction A reagent for the cleavage reaction including a solution may be included.   The method of the present invention allows simultaneous analysis of both strands (e.g., sense and antisense strands). , Ideal for high level multiplexing. The products obtained are qualitative and quantitative Easy to adapt to location analysis. The method may be automated, and Is It may be performed in a solid phase (eg, on a solid support). The method has a longer core than current methods. It is effective in allowing analysis of acid fragments.   Description of drawings   FIG. 1 shows that Thermus aquaticus (SEQ ID NO: 1) Thermus flavus (SEQ ID NO: 2) and Thermus thermofilus (T hermus thermophilus) (SEQ ID NO: 3) This is a comparison of the memory structures. The consensus sequence (SEQ ID NO: 7) is shown at the top of each column.   FIG. 2 shows that Thermus aquaticus (SEQ ID NO: 4) Thermus flavus (SEQ ID NO: 5) and Thermus thermofilus (T hermus thermophilus) (SEQ ID NO: 6). A column comparison. The consensus sequence (SEQ ID NO: 8) is shown at the top of each column.   FIG. 3 shows a CFLP that produces a characteristic fingerprint from a nucleic acid substrate.^TMThe law FIG.   FIG. 4 shows the organization of the human p53 gene. Exons are represented by solid black squares. Label as ~ 11. The five hot spot areas are the areas including exons 5 to 8 Shown as enlargements, the hot spot areas are labeled A, A ', B, C and D. Understand.   FIG. 5 shows the first two stages P for the generation of a DNA fragment containing the p53 mutation. FIG. 4 provides a diagram illustrating the use of CR technology.   FIG. 6 shows a second two-stage P for generation of a DNA fragment containing the p53 mutation. FIG. 4 provides a diagram illustrating the use of CR technology.   FIG. 7 shows a structure that cannot be amplified using DNAPTaq.   FIG. 8 shows that a branched double helix was formed using DNAPTaq or DNAPStf (Stoffel). 7 is an ethidium bromide stained gel showing an attempt to amplify.   FIG. 9 shows cleavage of branched double helix by DNAPTaq and DNAPStf. Figure 4 is an autoradiogram of a gel analyzing for loss of cleavage.   FIGS. 10A-B differ during attempts to cleave a branched duplex helix with DNAPTaq. Cleavage or cleavage when the reaction components are added and the incubation temperature is changed 1 is a set of autoradiograms of a gel analyzing the loss of DNA.   FIGS. 11A-B show timed with and without primers. It is an autoradiogram showing a cleavage reaction.   Figures 12A-B show an attempt to cleave a branched double helix with various DNAPs (primer primers). Auto-radiogram of a set of gels with and without is there.   FIG. 13A shows specific cleavage of the substrate DNA targeted by the leading oligonucleotide. 2 shows the substrates and oligonucleotides used to test the.   FIG. 13B shows a cleavage reaction using the substrate and the oligonucleotide shown in FIG. 13A. 5 shows an autoradiogram of the gel showing the results of the above.   FIG. 14A shows specific cleavage of the substrate RNA targeted by the lead oligonucleotide. 2 shows the substrates and oligonucleotides used to test the.   FIG. 14B shows a cleavage reaction using the substrate and the oligonucleotide shown in FIG. 14A. 5 shows an autoradiogram of the gel showing the results of the above.   FIG. 15 is a diagram of the vector pTTQ18.   FIGS. 16A-G are a set of diagrams of wild-type and synthetic defective DNAPTaq genes. .   FIG. 17 is a diagram of the vector pET-3c.   FIG. 18A shows the wild-type Thermus flavus polymerase gene. Is shown.   FIG. 18B shows a synthetic defective Thermus flavus polymerase gene. Indicates a child.   Figures 19A-E show suitable substrates for cleavage of DNAP by 5 'nuclease activity. Shows a set of molecules.   FIG. 20 is a gel autograph showing the results of the cleavage reaction performed using the synthesis-deficient DNAP. It is a tradogram.   FIG. 21 shows the assay products of the synthetic activity in the synthesis-deficient DNAPTaq clone. It is an autoradiogram of a PEI chromatogram separated.   FIG. 22A tests the ability of synthetic deficient DNAP to cleave short hairpin structures. The substrate molecule used for the measurement is shown.   FIG. 22B separates the products of the cleavage reaction performed using the substrate shown in FIG. 22A. 3 shows an autoradiogram of a gel.   FIG. 23 shows the complete 206 mer (206-mer) used as a substrate for the 5 ′ nuclease of the present invention. mer) Provides a double helix sequence.   FIGS. 24A and 24B show Thermus aquaticus and Wild-type DNAP isolated from Thermus flavus and 5 ' FIG. 9 shows the cleavage of a linear nucleic acid substrate (based on the 206 mer in FIG. 23) by a nuclease. You.   FIG. 25A shows the “nibbling” phenomenon detected with the DNAP of the present invention. Show.   FIG. 25B shows that “nibbling” in FIG. Not 5 'nucleotide cleavage.   FIG. 26 shows that the "nibbling" phenomenon is double helix dependent. Show. FIG. 27 shows MgCl_TwoOr MnCl_TwoOf the cleavage reaction performed in the presence of 1 shows an autoradiograph of a gel for separating S.   FIG. 28 shows the generation of a cleavage reaction performed on four similarly sized DNA substrates. Fig. 3 shows an autoradiograph of a gel separating substances.   FIG. 29 shows the results obtained using wild-type and two mutant tyrosinase gene substrates. 2 shows an autoradiograph of a gel that separates the products of the cleaved cleavage reaction.   Figure 30 shows various lengths of wild-type or protruding from 157 nucleotides to 1.587 kb. Isolate the products of cleavage reactions performed with any of the mutated tyrosinase substrates 3 shows an autoradiograph of a gel.   FIG. 31 shows various concentrations of MnCl._TwoTo separate the products of the cleavage reaction performed in 3 shows an autoradiograph of the radiator.   FIG. 32 shows gels that separate the products of cleavage reactions performed in various concentrations of KCl. 3 shows an autoradiograph.   FIG. 33 shows the gel auto-separation product of the cleavage reaction performed at various reaction times. 3 shows a radiograph.   FIG. 34 shows the autoradiography of the gel separating the products of the cleavage reaction performed at various temperatures. 3 shows an ograph.   FIG. 35 shows various amounts of the enzyme Cleavase.^TMCleavage performed with BN Figure 4 shows an autoradiograph of a gel separating the products of the reaction.   FIG. 36 shows the products of cleavage reactions performed using four different preparations of DNA substrate. 1 shows an autoradiograph of a gel for separating S.   FIG. 37 shows the sense or antisense of four different tyrosinase gene substrates. Figure 4 shows an autoradiograph of a gel separating the products of the cleavage reaction performed on the chains.   FIG. 38 shows wild-type β-blobi at four different temperatures in two different concentrations of KCl. FIG. 4 shows an autoradiograph of a gel separating the products of a cleavage reaction performed on a substrate. .   FIG. 39 shows two different mutant β-brobin groups in five different concentrations of KCl. Figure 4 shows an autoradiograph of a gel separating the products of the cleavage reaction performed on the mass.   FIG. 40 shows cleavage reactions performed on wild-type and three mutant β-brobin substrates. 5 shows an autoradiograph of a gel separating the products of Example 1.   FIG. 41 is an autograph of a gel separating the products of cleavage reactions performed on RNA substrates. 3 shows a geograph.   FIG. 42 shows the enzyme Cleavase.^TMBN or Taq DNA polymerase Is used as a 5 'nuclease to separate the products of cleavage reactions 3 shows an autoradiograph of a gel.   FIG. 43 shows the cleavage reaction performed on double-stranded DNA substrate to show the multiplexing of the cleavage reaction. 3 shows an autoradiograph of a gel separating the corresponding products.   FIG. 44 shows 419 and 42 derived from exon 4 of the human tyrosinase gene. Various concentrations of MnCl on a double-stranded DNA substrate consisting of two mutant alleles_TwoDuring ~ 5 shows an autoradiograph of a gel for separating a product of the cleavage reaction performed in the step (a).   FIG. 45 shows two differently labeled substrates (exons of the tyrosinase gene). Cleavage resulting from a cleavage reaction involving the wild type and 422 mutant substrates from Represents the two channel signals (JOE and FAM fluorescent dye) for the fragment Two records are shown. The thin line represents the JOE-labeled wild-type substrate, and the thick line represents FAM-labeled 4 Represents the 22 mutant substrate. On the record, it is derived from exon 4 of the tyrosinase gene. Performed on double-stranded DNA substrates consisting of the wild-type and 422 mutant alleles 2 shows an autoradiograph of a gel that separates the products of the cleaved cleavage reaction.   FIG. 46 shows the nuclei of the six SIVLTR clones corresponding to SEQ ID NOs: 63-68. Shows the reotide sequence.   FIG. 47 shows six different duplexes S containing a biotin label at the 5 ′ end of the (−) strand. Autoradiographic gel separation of the products of cleavage reactions performed on IVLTR substrates Shows   Figure 48 shows six different double stranded S containing a biotin label at the 5 'end of the (+) strand. Autoradiographic gel separation of the products of cleavage reactions performed on IVLTR substrates Shows   FIG. 49 isolates the products of single-strand cleavage reactions performed in various concentrations of NaCl. 1 shows an autoradiograph of a gel.   FIG. 50 shows various concentrations of (NH_Four)_TwoSO_FourThe product of the single-strand cleavage reaction performed in 4 shows an autoradiograph of a gel to be separated.   FIG. 51 isolates the products of single-strand cleavage reactions performed in increasing concentrations of KCl. 1 shows an autoradiograph of a gel.   FIG. 52 shows the results of single-strand cleavage reactions performed in KCl at two concentrations and at various reaction times. Figure 3 shows an autoradiograph of a gel separating the products.   FIG. 53 shows the products of cleavage reactions performed on the same substrate in single-stranded or double-stranded form. 4 shows an autoradiograph of a gel to be separated.   FIG. 54 isolates the products of double-strand cleavage reactions performed in various concentrations of KCl. 3 shows an autoradiograph of a gel.   FIG. 55 isolates the products of double-strand cleavage reactions performed in various concentrations of NaCl. 1 shows an autoradiograph of a gel.   FIG. 56 shows various concentrations of (NH_Four)_TwoSO_FourThe product of the double-strand cleavage reaction performed in 4 shows an autoradiograph of a gel to be separated.   FIG. 57 shows gels separating the products of double-strand cleavage reactions performed at various reaction times. 3 shows an autoradiograph.   FIG. 58 shows various amounts of Cleavase for 5 seconds or 1 minute.^TMBN yeast Autoradiograph of a gel separating the products of a double-strand cleavage reaction performed with iodine Is shown.   FIG. 59 shows gel gels separating the products of double-strand cleavage reactions performed at various temperatures. Shows a tradograph.   FIG. 60 shows various amounts of Cleavase.^TMTwo using BN enzyme Figure 4 shows an autoradiograph of a gel separating the products of the chain cleavage reaction.   FIG. 61A shows the products of a single-strand cleavage reaction performed in buffers with different pH. 4 shows an autoradiograph of a gel to be separated.   FIG. 61B shows one run in a buffer having a pH of either 7.5 or 7.8. 3 shows an autoradiograph of a gel separating the products of this strand cleavage reaction.   FIG. 62A shows two runs performed in a buffer having a pH of either 8.2 or 7.2. 3 shows an autoradiograph of a gel separating the products of this strand cleavage reaction.   FIG. 62B shows that two runs were performed in a buffer having a pH of either 7.5 or 7.8. 3 shows an autoradiograph of a gel separating the products of this strand cleavage reaction.   FIG. 63 shows the results of single-strand cleavage reactions performed in the presence of various amounts of human genomic DNA. 1 shows an autoradiograph of a gel separating the product.   FIG. 64 was performed using two different concentrations of Tfl DNA polymerase in KCl. 1 shows an autoradiograph of a gel separating the products of the single-strand cleavage reaction.   FIG. 65 shows the results obtained using two different concentrations of Tth DNA polymerase in KCl. 1 shows an autoradiograph of a gel separating the products of the single-strand cleavage reaction.   FIG. 66 shows the use of E. coli Exo III enzyme in KCl at two different concentrations. Figure 4 shows an autoradiograph of a gel separating the products of the performed single-strand cleavage reactions.   FIG. 67 shows three different tyrosinase gene substrates (SEQ ID NOs: 34, 41 and 42) Upper Tth DNA polymerase, E. coli Exo III enzyme or Cleavase^TMIsolate the products of single-strand cleavage reactions performed with any of BN 1 shows an autoradiograph of a gel.   FIG. 68 is a drawing showing the positions of the 5 ′ and 3 ′ cleavage sites on the cleavage structure.   FIG. 69 shows three different tyrosinase gene substrates (SEQ ID NOs: 34, 41 and 42) Upper Cleavase^TMUse either BN or Rad1 / Rad10 complex Shows an autoradiograph of the gel separating the products of the single-strand cleavage reaction performed .   FIG. 70 shows double-stranded cleavage performed on wild-type and two mutant β-globin substrates. Figure 4 shows an autoradiograph of a gel separating the products of the cleavage reaction.   FIG. 71A shows single strands performed on wild-type and three mutant β-globin substrates. Figure 4 shows an autoradiograph of a gel separating the products of the cleavage reaction.   FIG. 71B shows the results of single-strand cleavage reactions performed on five mutant β-globin substrates. 1 shows an autoradiograph of a gel separating the product.   FIG. 72 shows the products of the double-strand cleavage reaction in which the order of addition of the reaction components varies. 4 shows an autoradiograph of a gel to be separated.   FIG. 73 shows the generation of cleavage reactions performed on wild-type and two mutant p53 substrates. Fig. 3 shows an autoradiograph of a gel separating substances.   FIG. 74 shows the generation of cleavage reactions performed on wild-type and three mutant p53 substrates. Fig. 3 shows an autoradiograph of a gel separating substances.   FIG. 75 shows the cleavage reaction performed on wild-type and mutant p53 substrates, The products of the cleavage reaction where the mutant and wild-type substrates are present at various concentrations relative to each other. 1 shows an autoradiograph of a gel for separating S.   In FIG. 76, HCV clone 1.1 (SEQ ID NO: 108) and HCV 2.1 (SEQ ID NO: 108) 109), HCV 3.1 (SEQ ID NO: 110), HCV 4.2 (SEQ ID NO: 111), HC V6.1 (SEQ ID NO: 112) and HCV 7.1 (SEQ ID NO: 113) provided in alignment I do.   FIG. 77 shows six double-stranded HCV groups labeled on the sense or antisense strand. Scan of a gel to separate the products of cleavage reactions performed on a sample (fluoroimager scan).   FIG. 78 shows wild-type and two mutant M. tuberculosis rpoB groups. 1 shows an autoradiogram of a gel separating the products of the cleavage reaction performed on the mass.   FIG. 79A shows wild-type and dTTP or dUTP prepared using either dTTP or dUTP. Of cleavage reactions performed on two mutant M. tuberculosis rpoB substrates Perform a fluoroimager scan of the gel to separate the products. Show.   FIG. 79B shows the fluoroimage of the gel shown in FIG. 85A after longer electrophoresis. 2 shows a jar scan (fluoroimager scan).   FIG. 80 shows wild-type and three mutant M. tuberculosis (M. .tuberculosis) Automated gel to separate the products of cleavage reactions performed on katG substrate 3 shows a radiogram.   FIG. 81 shows wild-type and three mutant human ligation labeled on the antisense strand. Gels that separate the products of cleavage reactions performed on M. tuberculosis katG substrate 1 shows a fluoroimager scan.   FIG. 82 shows a plasmid along the sequence of the E. coli rrsE gene (SEQ ID NO: 145). Indicates the position of the immer.   In FIG. 83, E. coli rrsE (SEQ ID NO: 145), Campylobacter di Jejuni5 (SEQ ID NO: 146) and Staphylococcus aureu Aureus (SEQ ID NO: 147) rRNA gene (common PCR rRNA primer Key positions are shown in bold).   FIG. 84 isolates the products of cleavage reactions performed on four bacterial 16S rRNA substrates 1 shows a fluoroimager scan of a running gel.   FIG. 85A shows the products of cleavage reactions performed on five bacterial 16S rRNA substrates. Figure 4 shows a fluoroimager scan of the gel to be released.   FIG. 85B shows the products of cleavage reactions performed on five bacterial 16S rRNA substrates. Figure 5 shows a bacterial fluoroimager scan of the gel to be released.   FIG. 86 isolates the products of cleavage reactions performed on various bacterial 16S rRNA substrates. 1 shows a bacterial fluoroimager scan of a running gel.   FIG. 87 isolates the products of cleavage reactions performed on eight bacterial 16S rRNA substrates. 1 shows a bacterial fluoroimager scan of a running gel.   FIG. 88 shows naturally occurring deoxynucleotides or deoxynucleotides. Performed on wild-type and mutant tyrosinase gene substrates prepared using analogs 5 shows an autoradiogram of a gel that separates the products of the cleavage reaction.   Definition   To facilitate understanding of the invention, some terms are defined below.   The term "gene" refers to the regulatory sequences and necessary sequences necessary for the production of a polypeptide or precursor. And a DNA sequence comprising the coding sequence. The polypeptide is Only by full-length coding sequence or if desired enzyme activity is retained It can be encoded by any part of the coding sequence.   The term "wild-type" refers to a gene or gene when isolated from a naturally occurring source or A gene or gene product having the characteristics of a gene product is meant. Wild-type gene Is most frequently observed in a population and therefore the "normal" or It is referred to as "wild type" without permission. In contrast, "modification ( The term `` modified '' or `` mutant '' refers to a wild-type gene or gene product. When compared, a modification of a sequence and / or a modification of a functional property (ie, an altered Gene or gene product exhibiting the above properties). Naturally occurring mutants Can be isolated, and these can be compared to wild-type genes or gene products. It is noteworthy that they are identified by the fact that they have altered properties.   As used herein, the term “recombinant DNA vector” refers to a desired coding sequence. Coding sequence and said coding sequence operably linked in a particular host organism Means a DNA sequence containing an appropriate DNA sequence necessary for the expression of In prokaryotes DNA sequences required for expression in E. coli include a promoter, if necessary, an operator Sequences, ribosome binding sites and possible other sequences. Eukaryotic cells Use of promoters, polyadenylation signals and enhancers Have been.   As used herein, the term "LTR" refers to provirus (ie, retrovirus). (Integrated form of the virus). The LT R is a transcription factor, polyadenylation signal, and replication of the viral genome And contains a number of regulatory signals including sequences required for integration. The virus L TR is divided into three regions called U3, R and U5.   The U3 region contains enhancer and promoter elements. U5 area Contains a polyadenylation signal. R (repeat) region is U3 region and U5 region And the transcribed sequence of the R region appears at both the 5 ′ and 3 ′ ends of the viral RNA. I do.   The term "oligonucleotide" as used herein is more than one, preferably Is 3 or more, usually 10 or more deoxyribonucleotides or ribonucleotides. Is defined as a molecule consisting of The exact size depends on many factors, and these factors The child in turn depends on the ultimate function or use of the oligonucleotide. The oligonu Nucleotides include any, including chemical synthesis, DNA replication, reverse transcription or a combination thereof. It may be generated by any of these methods.   Mononucleotides have the 5 'phosphate group of one mononucleotide pentose ring. An embodiment in which the 3 'oxygen adjacent thereto is bonded in one direction via a phosphodiester bond To produce an oligonucleotide, the end of the oligonucleotide, The 5 'phosphate group is not attached to the 3' oxygen of the mononucleotide pentose ring. Et al., The "5 'end" and the 3' oxygen of the 5 'end of the subsequent mononucleotide pentose ring. If not attached to the acid group, it is called the "3 'end". In this specification, even if the nucleus Even if the acid sequence is inside a larger oligonucleotide, Sometimes referred to as having 'and 3' ends.   Two different non-overlapping oligonucleotides having the same linear complementary nucleic acid sequence Annealed to different regions of one of the oligonucleotides, the 3 ' When facing the other 5 'end, the former is the "upstream" oligonucleotide and the latter is the "upstream" oligonucleotide. Sometimes referred to as "downstream" oligonucleotides.   The term "primer", when placed under conditions where primer extension begins, An oligonucleotide capable of acting as a starting point for synthesis is meant. Oligo Nucleotide `` primers '' may be present naturally as purified restriction digests. Alternatively, it may be generated synthetically.   Primers should be "essentially" complementary to the strand of the template's specific sequence. To be elected. The primer is hybridized with the template strand so that primer extension occurs. Must be complementary enough to form. The primer sequence is the template Need not reflect the exact sequence of For example, if a non-complementary nucleotide fragment It may be attached to the 5 'end of the primer, in which case the remaining primer sequence The other portion is essentially complementary to the strand. Hybridization, and it Sufficient to form a template-primer complex for the synthesis of primer extension products Each time the primer sequence has complementarity with the template sequence, Alternatively, longer sequences may be interspersed in the primer.   The “hybridization” method involves complementary arrangement with the target nucleic acid (the sequence to be detected). Includes column annealing. Two nucleic acid polymers containing complementary sequences are base It is well recognized that pairing interactions can find and anneal to each other. That is the phenomenon. Marmur and Lane (Proc. Natl. Acad. Sci. USA 46: 453 (196 0)) and Doty et al. (Proc. Natl. Acad. Sci. USA 46: 461 (1960)). After the first observations of the "ad formation" process, the process became more sophisticated and It is an indispensable means. Nevertheless, on some issues More extensive use of hybridization as a tool in human diagnostics Is hindered. Particularly difficult problems are: 1) Hybridization Inefficient 2) Low concentration of specific target sequence in genomic DNA mixture And 3) only some of the complementary probes and targets hybridize That.   In terms of efficiency, only a small fraction of the possible number of probe-target It has been observed experimentally that it is not formed during the metal-forming reaction. This is a short cage This is especially true for oligonucleotide probes (less than 100 bases in length). This is Due to three root causes: a) Hybridization due to secondary and tertiary structure interactions B) the DNA strand containing the target sequence is Rehybridization (re-annealing), and c) some target molecules When used in a hybridization format where the target nucleic acid is immobilized on a solid phase, Hybridization is inhibited.   If the probe sequence is completely complementary to the target sequence (i.e., the primary structure of the target) Even so, does the target sequence approach the probe via higher order rearrangements? I have to. The rearrangement of these higher-order structures may be due to the secondary or triad of the molecule. It may be involved in any of the following structures: Secondary structure is determined by intramolecular bonds Is determined. For DNA or RNA targets, this may be a high within a single contiguous base strand. Consists of bridging (as opposed to hybridization between two different strands). molecule Depending on the degree and location of internal binding, the probe separates from (displaces) the target sequence. , Hybridization can be inhibited.   Hybrid forms of oligonucleotide probes for denatured double-stranded DNA in liquid Synthesis is more complicated because longer complementary target strands can be reconstituted or rear-annealed. is there. Again, the hybridized probe is detached in this process ( Replaced). This results in a hybrid to the starting concentration of probe and target The yield of formation is low (low coverage).   Regarding the low target sequence concentration, the DNA fragment containing the target sequence is usually And its abundance in genomic DNA is relatively low. This shows great technical difficulty And the most common method using oligonucleotide probes is It lacks the sensitivity required to detect hybridization at such low levels.   One attempt to solve this target sequence concentration problem involves amplification of the detection signal. It is. Most often, this involves using one or more labels with oligonucleotide probes. Need to be placed on top. Highest affinity for non-radioactive labels -Single copy in genomic DNA using oligonucleotide probes, even reagents It has been found to be unsuitable for gene detection. Wallace et al., Biochimie 67: See 755 (1985). For radioactive oligonucleotide probes, very Only high specific activity has been found to give satisfactory results. Studencki and W See allace, DNA 3: 1 (1984) and Studencki et al., Human Genetics 37:42 (1985). It was done.   With respect to complementation, in some diagnostic applications, hybridization is a complete phase. It is important to determine whether it exhibits complementarity or partial complementarity. For example, disease Presence of original DNA (eg, from viruses, bacteria, fungi, mycoplasmas, protozoa) If simple detection of presence or absence is desired, hybridization methods Is only important to ensure hybridization (if related sequences are present). When partially complementary and fully complementary probes hybridize together Conditions can be selected). However, in other diagnostic applications, the Hybridization may need to distinguish between partial and complete complementarity . You may be interested in detecting genetic polymorphisms. For example, human hemog Robin is partially composed of four polypeptide chains. Two of these chains are 141 Of the same amino acid (α chain), two of which are 146 amino acids (β chain) Are the same chains. Genes encoding beta chains are known to exhibit polymorphism . The normal allele encodes a beta chain with glutamic acid at position 6. Sudden change The heteroallele encodes a beta chain with a valine at position 6. This amino acid difference Meaningful (mutant allele known clinically as sickle cell anemia) (The most significant if the individual is homozygous). Ami On the genetic basis of the acid changes, the normal allele DNA sequence and the mutant allele DN It is well known that single base differences from the A sequence are involved.   Allows the same level of hybridization in both partial and full complementarity Methods that are typically combined with other techniques (e.g., restriction enzyme analysis) Is inappropriate for such an application. The probe has both normal and mutant target sequences. Will hybridize to Hybridization is related to the method used. The sequence being assayed (target sequence) and the DNA fragment used to perform the test (Probe) requires some degree of complementarity. (Of course, there is no complementarity No binding can be obtained, but this binding is non-specific and should be avoided It is. )   As used herein, the complementarity of a nucleic acid sequence is such that the 5 'end of one sequence is 3' to the other. Oligos that are "anti-parallel" when aligned with the nucleic acid sequence to match the ends Means nucleotide. Certain bases not commonly found in natural nucleic acids May be contained in the nucleic acid of, for example, inosine, 7-deazaguanine and the like. No. Complementarity need not be perfect; stable double helices form mismatched base pairs Alternatively, it may contain an unpaired base. For those skilled in the nucleic acid art, Oligonucleotide length, oligonucleotide base composition and sequence, Considering a number of variables, including the strength of mismatches and the frequency of mismatched base pairs, The qualitative can be determined empirically.   The stability of a nucleic acid duplex is measured by its melting temperature, ie, "Tm". Can be. Under certain conditions, the Tm of a particular nucleic acid duplex is on average half a base pair. The temperature at which a minute dissociates.   The term "probe," as used herein, refers to a double helix with a sequence in another nucleic acid. Forming a structure (with at least one of the sequences in the probe nucleic acid (Due to sequence complementarity) and labeled oligonucleotides.   The term `` label '' as used herein is detectable (preferably quantifiable) Can be used to obtain a signal and bind to nucleic acids or proteins Means any atom or molecule. Labels are fluorescence, radioactivity, colorimetric Analysis, gravimetric analysis, X-ray diffraction or absorption, magnetism, enzyme activity, etc. It may give a signal.   As used herein, the term "cleavable structure" refers to a single-stranded nucleus containing a secondary structure. A region of an acid substrate that can be used as a cleavage means, including but not limited to enzymes. Means more cleavable. Regardless of secondary structure (i.e., substrate folding) (Without the need for folding) by agents such as phosphodiesterases that cleave nucleic acid molecules. In contrast to nucleic acid molecules that are substrates for non-specific cleavage, the cleavage structure Substrate for specific cleavage by the step.   The term "cleaving means" as used herein has the ability to cleave a cleavage structure. Means, for example, an enzyme, but is not limited thereto. Not. As the cleavage means, natural DNAP having 5 'nuclease activity ( For example, Taq DNA polymerase, E. coli DNA polymerase I), more details Specifically, there are modified DNAPs having 5 'nuclease activity but lacking synthetic activity. I can do it. Cleavage of naturally occurring structures in nucleic acid templates (structure-specific cleavage) The ability of the 5 'nuclease to determine the specific sequence in the nucleic acid in advance And is useful for detecting internal sequence differences in nucleic acids. In this way, they are It is a production specific enzyme. Structure-specific enzymes recognize specific secondary structures in nucleic acid molecules. Are enzymes that cleave these structures. The cleavage site extends to the 5 'side of the cleavage structure. Or on the 3 ′ side, or alternatively, the cleavage site is 5 ′ of the cleavage structure. Between the side and the 3 ′ side (ie, inside the cleavage structure). Opening of the present invention The cleavage means cleaves the nucleic acid molecule in response to the formation of a cleavage structure. The cleavage means is Cleavage of the cleavage structure at any particular position in the cleavage structure is not necessarily Not necessary.   The cleavage means is not limited to an enzyme having 5 'nuclease activity . The cleaving means is Cleavase.^TM, Taq DNA polymerase, colon E. coli DNA polymerase I and eukaryotic structure-specific endonuclease , Mouse FEN-1 endonuclease [Harrington and Liener, (1994) Genes a nd Develop. 8: 1344] and calf thymus 5 'to 3' exonuclease [Murante, R.S. et al., (1994) J.R. Biol. Chem. 269: 1191] It may contain a lease activity. In addition, DNA repair endonuclease Millie members (eg, from Drosophila melanogaster) RrpI enzyme, yeast RAD1 / RAD10 complex and 3 ′ nuclei such as E. coli Exo III) Enzymes having creatase activity are also suitable cleavage means for carrying out the method of the invention.   As used herein, the term "cleavage product" refers to a means of cleaving reacting with a cleaved structure. (I.e., treating the cleavage structure with cleavage means) Means product.   The terms "nucleic acid substrate" and "nucleic acid template" are used interchangeably herein and And regenerated (i.e., folded by the formation of intrachain hydrogen bonds), A nucleic acid molecule that forms at least one cleavage structure. The nucleic acid substrate comprises It may be composed of single-stranded or double-stranded DNA or RNA.   The term "essentially single-stranded" as used in reference to a nucleic acid substrate refers to the It means that it exists as a single-stranded nucleic acid, and joins together by intrachain base-pairing interaction In contrast to double-stranded substrates that exist as two nucleic acid strands.   Nucleic acids form secondary structures that depend on base pairing for stability. Single-stranded nucleus Acids (single-stranded DNA, denatured double-stranded DNA or RNA) are used to separate different sequences (and When folded on their own (with a contiguous array), they have a characteristic Takes secondary structure. At “elevated temperature”, the double helix region of the structure follows the edge of instability. Healing results in maximizing the effect of small changes in the sequence and Appear as change. That is, the "elevated temperature" is a constant The temperature is around the temperature at which the double helix in the double helix region melts. Then In addition, the alteration of the substrate sequence results in the destruction of the double helix region, Different cleavage patterns may be obtained if dependent cleavage agents are used in the reaction. Would. Without being limited to any particular theory, the target (i.e., substrate) Each individual molecule in the group has only one or several potential cleavage structures (such as Double helix region), but the sample was analyzed as a whole. In this case, a composite pattern indicating all cleavage sites is detected. Active cleavage part Many of the structures recognized as positions are thought to be only a few base pairs long, It is considered unstable when elevated temperatures are used during the cleavage reaction. Or maybe Nevertheless, due to the transient formation of these structures, the cleavage means Recognition and cleavage are possible. The formation of these structures in response to small sequence changes Or the fracture alters the cleavage pattern. Temperatures in the range of 40-85 ° C (55- (A range of 85 ° C. is particularly preferred.)   As used herein, the term “sequence variation” refers to the sequence of a nucleic acid sequence between two nucleic acid templates. Means the difference. For example, the wild-type structural gene and the sudden Mutant forms may be single base substitutions and / or deletions or insertions of one or more nucleotides. The sequence may change due to the presence of the input. These two forms of the structural gene It is said that the arrangement changes with each other. Second mutant form of the structural gene Condition may exist. This second mutant form comprises the wild-type gene and the gene It is said that the sequence changes from both of the first mutant form. However In the present invention, it is necessary to compare between one or more gene forms to detect a sequence variation. It is noted that there is no need. The method of the invention is characteristic for a given nucleic acid substrate. Relevant to wild-type or other controls to give reproducible cleavage product patterns Alternatively, a characteristic "fingerprint" can be obtained from any nucleic acid substrate. Book The invention "fingerprints" the nucleic acid independently of the control, and By comparing the mutant form with a wild-type or known mutant control, Use of the method to identify a mutated form of the acid is contemplated.   The term "releasing" as used herein is due to the action of 5 'nuclease. Release nucleic acid fragments from larger nucleic acid fragments (e.g., oligonucleotides) In which case the free fragments are no longer present in the remainder of the oligonucleotide. Not covalently bonded.   The term “substrate strand” as used herein is mediated by 5 ′ nuclease activity Means the nucleic acid strand in the cleavage structure in which the cleavage to be performed occurs.   The term "template strand" as used herein refers to at least a portion of the substrate strand. Are complementary to each other and anneal to the substrate strand to form a cleavage structure; It means the nucleic acid chain in the cleavage structure.   As used herein, the term "Km" refers to the Michaelis Means the maximum rate of the enzyme in an enzyme-catalyzed reaction. It is defined as the concentration of the specific substrate that gives a factor of one.   As used herein, the term “nucleotide analog” refers to 7-deazapurine ( That is, modified such as 7-deaza-dATP and 7-deaza-dGTP) Or non-naturally occurring nucleotides. Salts for nucleotide analogs Modified forms of deoxyribonucleotides and ribonucleotides, including group analogs State. The present invention relates to the substrate present in the nucleic acid amplification mixture (e.g., PCR mixture). The term "nucleotide analog" as used in the textbook refers to dATP, dGTP, implies the use of nucleotides other than dCTP and dTTP, The use of dUTP (naturally occurring dNTP) in R Including the use of analogs. dUTP, 7-deaza-dATP, 7-deaza-dGTP Or any other nucleotide analog was used in the reaction mixture The PCR product is said to contain a nucleotide analog.   "Oligonucleotide primers that match or are complementary to the gene sequence" Oligonucleotides capable of promoting template-dependent synthesis of single- or double-stranded nucleic acids Reotide primer is meant. Oligones that match or are complementary to the gene sequence Nucleotide primers may be used in PCR, RT-PCR, and the like.   A "common gene sequence" is derived by comparing two or more gene sequences. A gene sequence representing the nucleotide most frequently present in a certain segment of the gene Means column. The consensus sequence is a defined sequence.   The term “polymorphic locus” is used in a population where mutations occur among members of the population. (Ie, the most common allele is less than 0.95) With frequency). In contrast, “monomorphic loci” are seen among members of the population. Loci with few or no mutations (in the gene pool of the population) Are generally considered to be loci with a frequency greater than 0.95 ing).   As used herein, the term "microorganism" refers to a microorganism that is too small to be observed with the naked eye. Substance, including but not limited to bacteria, viruses, protozoa, fungi and ciliates It is not limited.   The term "microbial gene sequence" refers to a gene sequence derived from a microorganism.   The term “sequence from one or more microorganisms” refers to one or more microorganisms. A nucleic acid sequence extracted from a mixture of organisms. An opening comprising the nucleic acid sequence of the microorganism. Prior to processing to form and then cleave the cleaved structure, the extracted sequence It may be subjected to a treatment such as amplification (eg, polymerase chain reaction).   The term "bacteria" refers to any bacterial species, including eubacteria and archaea. To taste.   The term "virus" is not capable of autonomous replication (i.e., (Requires use of the device) means oblique ultramicroscopic intracellular parasites.   The term "multidrug resistance" refers to two or more antibiotics or antimicrobial agents used to treat microorganisms. A microorganism that is resistant to a microbial agent is meant.   As used herein, “CFLP^TM(Cleavase)^TMFragment length polymorphism The term "sex) analysis" refers to i) denaturation of a nucleic acid strand, ii) cooling, or otherwise intrachain secondary. Iii) identify nucleic acids having an intrachain secondary structure in response to said structure. The nucleic acid substrate is cleaved with a cleaving agent that is aware and cleaves, whereby the collection of fragments characteristic of the nucleic acid substrate. Analysis of the products of the reaction giving the cleavage product (i.e., often by electrophoresis) Means Nucleic acid substrates that differ in sequence from a control or reference nucleic acid substrate are separated in this manner. When analyzed, it shows an altered expression of the fragment in the cleavage product pool, 1 illustrates the presence of the sequence differences between qualities.   Description of the invention   The present invention provides methods and compositions for treating nucleic acids, particularly nucleic acid sequences and arrangements. Methods and compositions for detecting and characterizing column changes.   The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. In particular, the present invention has 5 'nuclease activity without inhibiting nucleic acid synthesis ability. It relates to a cleavage enzyme.   The present invention shows a DNA synthesis activity modified from a natural thermostable DNA polymerase. 5 'nuclease derived from a thermostable DNA polymerase is provided. The polymerer 5 'nuclease activity is retained, but its synthetic activity is reduced? Or lost. Such 5 'nucleases, without inhibiting synthetic activity, It has the ability to catalyze the structure-specific cleavage of nucleic acids. Lack of synthetic activity during the cleavage reaction As a result, the nucleic acid cleavage product has a fixed size.   The novel properties of the polymerases of the present invention are the basis for methods for detecting specific nucleic acid sequences. To form This method relies on the amplification of the detection molecule, and does not It does not depend on the amplification of the target sequence itself as in the case of the target sequence detection method.   DNA polymerase (DNAP) [e.g., isolated from E. coli Or from a thermophilic bacterium of the genus Thermus] It is an enzyme that synthesizes NA chains. Some of the known DNAPs increase the synthesis activity of the enzyme. In addition, it contains related nuclease activity.   Some DNAPs remove nucleotides from the 5 'and 3' ends of the DNA strand [Kornberg, DNA Replication, W.C. H. Freeman and Co. , San Francisco, pp 127-139 (1980)]. These nuclease activities are usually These are called 5 'exonuclease activity and 3' exonuclease activity, respectively. I have. For example, the 5 'exonucleotide in the N-terminal domain of some DNAPs The activity of the enzyme is determined by the removal of RNA primers during lagging strand synthesis during DNA replication. And removal of damaged nucleotides during repair and repair. Some DNAPs, for example large Enterobacteriaceae (E. coli) DNA polymerase (DNAPEcl) is also proofread during DNA synthesis. It has 3 'exonuclease activity that causes loading (Kornberg, supra).   Thermus Aquatics (Th) called Taq DNA Polymerase (DNAPTaq) ermus aquaticus) has 5 'exonuclease activity. But lacks a functional 3 'exonuclease domain [Tindall and Kunkell Biochem. 27: 6008 (1988)]. Klenow fragment and Stoffel, respectively Derivatives of DNAP Ecl and DNAP Taq, called fragments, can be obtained by enzymatic manipulation or As a result of the genetic manipulation, the 5 'exonuclease domain is missing [Brutlag et al., Bioche m. Biophys. Res. Commun. 37: 982 (1969): Erlich et al., Science 252: 1643 (1991);  Setlow and Kornberg, J .; Biol. Chem. 247: 232 (1972)].   It has been reported that simultaneous synthesis is required for the 5 'exonuclease activity of DNAPTaq. [Gelfand, PCR Technology-Principles and Applications for DNA Amp lification (H.A. Erlich), Stockton Press, New York, p.19 (1989)]. DNA Among the 5 'exonuclease digestion products of PTaq and DNAPEcl, Nucleotides predominate, but also short oligonucleotides (less than 12 nucleotides) Can be observed, which indicates that these so-called 5 'exonucleases Implies that it can function as an endonuclease [Setlow, supra; Ho] lland et al., Proc. Natl. Acad. Sci. USA 88: 7276 (1991)].   In International Publication WO 92/06200, Gelfand et al. The preferred substrate for exonuclease activity is indicated to be substituted single-stranded DNA. A phosphodiester bond is formed between the substituted single-stranded DNA and the double-stranded DNA. Degradation, but in this case the preferred exonuclease cleavage site is a double helix It is a phosphodiester bond in the region. Therefore, it is usually associated with DNAP. 5 ′ exonuclease activity is a structure-dependent single-stranded endonuclease, More suitably referred to as 5 'nuclease. Exonucleases are nucleic acid molecules It is an enzyme that cleaves a nucleotide molecule from its end. On the other hand, endonuclease , An enzyme that cleaves nucleic acid molecules not at the terminal site but inside. Some heat resistance D The nuclease activity associated with NA polymerase is endonuclease-wise. Cleavage, but this cleavage requires contact with the 5 'end of the molecule being cleaved. You. Therefore, these nucleases are called 5 'nucleases.   When 5 'nuclease activity is associated with eubacteria type A DNA polymerase , As independent functional domains, three minutes of the N-terminal region of the protein Found in one. Two-thirds of the C-terminus of the molecule is the polymer that initiates DNA synthesis. Configure the main. Also, some type A DNA polymerases have three minutes of the molecule. Has 3 'exonuclease activity associated with the two C-terminal regions.   The 5 'exonuclease activity and polymerization activity of DNAP were determined by the polymerase component. Offspring have been isolated by proteolytic cleavage or genetic manipulation. from now on, Remove or reduce 5 'nuclease activity while leaving polymerase activity intact The heat-resistant DNAP has been modified to reduce it.   DNA Klenow fragment or large proteolytic cleavage fragment of PEcl But has 5 'nuclease activity and 3' exonuclease activity. Lack. The Stoffel fragment of DNAPTaq (DNAPStf) is 5 ′ nucleus by genetic engineering to delete the N-terminal 289 amino acids Lack of lease activity [Erlich et al., Science 252: 1643 (1991)]. International Publication WO 92/062 No. 00 is a thermostable DN with an altered level of 5 'to 3' exonuclease. AP is described. U.S. Pat. No. 5,108,892 discloses a 5 'to 3' exonucleus. Description of the Thermus aquaticus DNAP without Zeze I have. However, in the field of molecular biology, heat-resistant D with a reduced amount of synthetic activity is used. Lacks NA polymerase.   The present invention relates to a method for retaining 5 'nuclease activity, but having reduced synthetic activity. Provides a 5 'nuclease from thermostable A-type DNA polymerase that is completely absent. It has already been reported that the synthesis activity of the enzyme can be uncoupled from the 5 'nuclease activity. As described (Gelfand, PCR Technology, supra), the 5 'nuclease activity It demonstrates that DNA synthesis is not required.   The description of the present invention can be found in I.D. Production of 5 'nuclease derived from thermostable DNA polymerase II. Cleavase for detection of secondary structure^TMFragment length polymorphism, II I. CFLP^TMDetecting mutations in the p53 tumor suppressor gene using the method And IV. CFLP^TMMethod is used to detect and identify pathogens.   I. Generation of 5 'nuclease from thermostable DNA polymerase   The method of the present invention uses a 5 'nuclease for detecting a specific nucleic acid sequence. The 5 ' The nuclease may be derived from a thermostable DNA polymerase. However The method of the present invention is not limited to the use of 5 'nuclease, Any cleavage that can generate a unique (i.e., characteristic) cleavage product pattern from Agents may be used. If you want to use 5 'nuclease, write As noted, the 5 'nuclease can be derived from a thermostable DNA polymerase. Good.   The genes encoding type A DNA polymerase are about one another at the DNA sequence level. Shares 85% homology. Preferred examples of thermostable polymerases include Thermus aquaticus, Thermus flav us), Thermus thermophilus and the like. I However, other thermostable A-type polymerases having 5 'nuclease activity are also suitable It is. 1 and 2 show the nucleotide sequences of the three polymerases and And compare the amino acid sequences. FIGS. 1 and 2 show the three heat-resistant DNA polymers. Common sequences obtained from comparison of nucleotide (FIG. 1) or amino acid (FIG. 2) sequences of the lase Alternatively, the majority sequence is shown in the top row. Amino acid residues in a given sequence These three polymers are identical if they are identical to those contained in the consensus amino acid sequence. A dot is placed in each sequence of the enzyme. To maximize alignment between display sequences, Use dashes to introduce gaps. Common nucleotchi at given position If no amino acids or amino acids are present, an "X" is added to the consensus sequence. Arrangement Column Nos. 1-3 show the nucleotide sequences of the three wild-type polymerases, Column numbers 4 to 6 show amino acid sequences. SEQ ID NO: 1 was isolated from strain YT-1 Wild-type Thermus aquaticus DNA polymerase gene [Lawyer et al., J. Am. Biol. Chem. 264: 6427 (1989)]. Sequence number 2 is for the wild-type Thermus flavus DNA polymerase gene Corresponding to the nucleic acid sequence [Akhmetzjanov and Vakhitov, Nucl. Acids Res. 20: 5839 ( 1992)]. SEQ ID NO: 3 is a wild-type Thermus thermophilu s) corresponding to the nucleic acid sequence of the DNA polymerase gene [Gelfand et al., International Publication WO 91 / 09950 (1991)]. SEQ ID NOs: 7 and 8 are common to the three DNAPs, respectively. The nucleotide and amino acid sequences are shown (also shown in the top row of FIGS. 1 and 2).   The 5 'nuclease of the present invention derived from a thermostable polymerase has reduced synthetic ability Retains essentially the same 5 'exonuclease activity as the native DNA polymerase You. As used herein, the term “essentially the same 5 ′ nuclease activity” refers to the term The 5 'nuclease activity of the modifying enzyme is changed to a structure-dependent single-stranded endonuclease. (But not necessarily the same rate of cleavage compared to the unmodified enzyme) It means retaining ability. Also has increased 5 'nuclease activity A-type DNA polymerases such that they produce enzymes with reduced levels of synthetic activity The protease may be modified. Reduced synthetic activity and increased 5 'nuclease activity Modified enzymes having the property are also contemplated by the present invention.   As used herein, the term “reduced synthetic activity” refers to a condition in which the modifying enzyme Synthetic activity levels below those found in decorative or "natural" enzymes Means to have. The modifying enzyme may not have any synthetic activity left, Or a synthetic activity that does not inhibit the use of the modified enzyme in the detection assays described below. May have a level. The 5 'nuclease of the present invention opens the polymerase. When cleavage activity is desired but synthesis ability is not desired (e.g., detection assays of the invention) Is the case).   As noted above, due to the nature of the modifications required to disrupt the polymerase synthesis, It is not intended that the invention be limited. The present invention relates to 1) proteolysis, 2) recombination Constructs (including mutants), 3) physical and / or chemical modifications and / or A variety of methods are contemplated, including but not limited to or inhibition.   1. Proteolysis   Physical cleavage of the unmodified enzyme with a proteolytic enzyme But by generating an enzyme fragment that retains 5 'nuclease activity, This produces a thermostable DNA polymerase with some level of synthetic activity. Tan After paq digestion, the resulting fragments are separated by standard chromatographic techniques And assay for the ability to synthesize DNA and act as a 5 'nuclease . The assay for measuring synthetic activity and 5 'nuclease activity is described below. Will be described.   2. Recombinant construct   In the following examples, 5 ′ nuclease derived from thermostable DNA polymerase was Preferred methods for producing constructs to be loaded are described. Type A DNA polymerase Because the DNA sequences are similar, Thermus aquaticus and The cloning strategy used for DNA and flavus polymerases was It is also applicable to type polymerases. Generally contains thermostable A-type DNA polymerase By isolating genomic DNA from bacteria that become infected using molecular biology methods, Clone the thermophilic DNA polymerase. This genomic DNA is The protease gene is exposed to primers capable of amplifying by PCR.   The amplified polymerase sequence is then subjected to standard deletion procedures to The polymerase portion of the offspring is deleted. A suitable deletion strategy is described in the examples below.   In the following examples, DNAPTaq polymer without loss of 5 'nuclease activity Discuss the strategy used to determine which parts of the protease domain can be removed. Deletion of amino acids from the protein may be due to mutation or frameshifting. Either by deleting the coding genetic material or by introducing a translation stop codon be able to. Further, to remove segments of the protein, the protein The protein can be subjected to proteolytic treatment.   In the examples below, the specific modification of the Taq gene is from nucleotide 1601 2502 (end of the coding region) deletion, 4 nucleotide insertion at position 2043 And nucleotides 1614 to 1848 and nucleotides 875 to 177 8 (numbering as in SEQ ID NO: 1). These modified sequences are: And SEQ ID NOs: 9-12.   One of skill in the art will recognize that a single base pair change would not harm the structure and function of the enzyme. to understand. Similarly, the exonuclease or polymerase function of these enzymes There can be small additions and deletions without essentially altering   Other deletions are also suitable for producing the 5 'nuclease of the present invention. The detection method of the present invention The use of 5 'nuclease in the assay to a level where synthetic activity does not inhibit Preferably, the deletion reduces the polymerase activity of the 5 'nuclease. No. Most preferably, the ability to synthesize is not present. For the assays described below Similarly, test modified polymerases for synthesis and presence of 5 'nuclease activity I do. A thorough study of these assays will allow for the screening of candidate enzymes of unknown structure. Leaning becomes possible. That is, the construct “ X "to determine that it is a 5 ' It can be determined whether or not a member of the genus creatase.   In the following examples, amplified Thermus aquaticus The PCR product of genomic DNA has the same nucleotide structure as natural genomic DNA. And did not have the same synthetic capacity as the original clone. Polymerase gene P Base pair changes caused by infidelity of DNAPTaq during CR amplification were also This is one method that can inactivate the ability to synthesize genes. Example below and FIG. A and 5A are natural Thermus aquaticus and Fra Shows that region in flavus DNA polymerase appears likely to be important for synthesis You. There are other base pair changes and substitutions that are likely to inactivate the polymerase.   However, a method for producing 5 'nuclease from DNA polymerase has been developed. It is not necessary to start with such a mutated amplification product. This is A method performed in the following example to obtain a DNA-deficient DNAPTaq mutant is described below. Out for the introduction of deletions, insertions and substitutions to produce 5 'nucleases One of skill in the art will understand that a wild-type DNA polymerase sequence may be used as a birthplace. It is. For example, to generate a synthetically deficient DNA PTfl mutant, SEQ ID NO: Thermus flavus using the primers according to 13-14 The wild-type DNA polymerase gene was amplified from the AT-62 strain. Then, the increase The width polymerase gene is subjected to restriction enzyme digestion to obtain a domain encoding the synthetic activity. Most of the amino acids were deleted.   It is contemplated by the present invention that the nucleic acid construct of the present invention is capable of expression in a suitable host. It is. Various promoters and 3 'sequences for efficient expression Methods for conjugation to are known to those skilled in the art. In the following example, two suitable vectors And six suitable vector constructs are disclosed. Of course, other suitable promotions There are also combinations of ter / vector. For expression of a nucleic acid construct of the present invention, a host It is not necessary to use things. For example, encoded by a nucleic acid construct The expression of the protein is performed by using a cell-free in vitro transcription / translation system. You may. One example of such a cell-free system is the commercially available TnT^TMConjugate reticulated red It is a blood cell lysate system (Promega Corporation, Madison, WI).   Once a suitable nucleic acid construct has been made, the 5 'nuclease may be produced from the construct. Good. According to the following examples and standard molecular biology teachings, different suitable methods Allows the construct to be manipulated.   Once the 5 'nuclease has been expressed, synthesis and nucleation are performed as described below. The polymerase is tested for both activities of ase. 3. Physical and / or chemical modification and / or inhibition   Synthetic activity of thermostable DNA polymerases is subject to chemical and / or physical means Therefore, it can be reduced. In one embodiment, the 5'- Cleavage reaction catalyzed by nuclease activity favors polymerase synthesis activity Operating under conditions that inhibit Synthetic activity level requires any significant synthetic activity Need to be reduced to a level that does not interfere with the cleavage reaction It is only.   Mg in concentrations greater than 5 mM as shown in the examples below^-By natural DNA The polymerization activity of PTaq is inhibited. 5'-nuclease under conditions where synthetic activity is inhibited The ability of the enzyme to function is a range of concentrations of Mg^-(5-10 mM) described below By manipulating assays for the activity of synthetic and 5'-nucleases Test. Predetermined concentration of Mg^-The effect of It is determined by quantifying the amount of synthesis and cleavage in the test reaction.   Other ions, polyamines, denaturants such as urea, formamide, dimethyl sulf Foxide, glycerin and nonionic surfactants (Triton X-100 and Tween-20) , Nucleic acid binding chemicals such as actinomycin D, ethidium bromide and sodium The inhibitory effect of ralen is the standard moderator for synthesis and 5'-nuclease assays. Tested by their addition to the spar. Next, the synthesis activity of thermostable polymerase 5′-nuclease activity (using compounds that have a preferential inhibitory effect on Cleavage) is maintained, while reducing or eliminating synthetic activity.   The use of physical means to preferentially inhibit the polymerase's synthetic activity it can. For example, the synthetic activity of thermostable polymerases (Typically 96-100 ° C) for prolonged exposure (20 minutes or more) Destroyed. There is only a small difference between each of the enzymes with respect to detailed heat tolerance. But they are easily measured. The polymerase is treated with heat for various periods of time, The effect of the heat treatment on the synthetic activity and the 5'-nuclease activity is determined. II. Cleavase for detection of secondary structure^TMFragment length polymorphism   Nucleic acids adopt a base-pair dependent secondary structure for stability. Even closely related Even if a single strand of nucleic acid having a different sequence (single-stranded DNA, denatured DNA or RNA) When they can fold themselves, they adopt a characteristic secondary structure. Construction These differences in the separation of DNA fragments with closely related sequences The ability to analyze single-stranded conformational polymorphism (SSCP) to perform   The 5'-nuclease domain of certain DNA polymerases has a sequence-specific Nuclear with a specific structure rather than expression (as does restriction endonucleases) It is a specific endonuclease that recognizes and cleaves acids. Described here DNAPTaq isolated nuclease domain (Cleavasase enzyme) e)^TM) Recognizes double-stranded ends with non-base-paired strands at the ends . Strands having a 5'-end are cleaved at the junction between the single and double strands.   FIG. 3 shows a wild-type substrate and a mutant substrate, wherein the mutant substrate has one base. Changes (changes from A to G as shown) differ from the wild type. In the method of the present invention According to the substrate structures, the nucleic acids can be denatured and fold on themselves. (See FIG. 3, steps 1 and 2). In the denaturation step, the nucleic acid is heat-treated, <3) or high (> 10) pH treatment, use of low salt concentration, cations, chemicals (eg, Achieved by the absence of urea, formamide) or proteins (eg helicase) Can be achieved. Nucleic acid folding or recombination can be caused by lowering the temperature, adding salts, neutralizing pH, Achieved by removal of chemicals or proteins.   The manner in which the substrate folds depends on the sequence of the substrate. 5'-Nuclear of the present invention Ze cleaves the structure. (See FIG. 3, Step 3). The end point of the generated fragment is cleaved Reflect the position of the part. The cleavage itself is not a specific sequence at the cleavage site, Depends on the formation of a particular structure.   When the 5'-nuclease of the present invention cleaves a nucleic acid substrate, the cleavage product or fragment Is generated. These fragments are characteristic fingerprints of nucleic acids that can be detected. [Eg, by electrophoresis on a gel (see step 4)]. (Eg, a single point mutation between the wild type and the mutant gene) Change the pattern of the cleaved structure. The 5'-nuclease of the present invention is a wild-type substrate And cleave structures formed by altered or mutated forms In this case, the distribution of the generated cleavage fragments differs between the two substrates, and the sequence of the two substrates (See FIG. 3, step 5).   Cleavase^TMEnzymes are unique cleavage product patterns for substrate nucleic acids Generate Detect single base changes in large length (eg, 1.6 kb) DNA molecules Cleavase for^TMUsing enzymatic digestion, the characteristic cleavage product Turns can be generated. The method of the present invention is based on "Cleavase".^TM Fragment length polymorphism (CFLP^TM) ". However, the present invention does not Alease^TMThe preferred enzymatic cleavage activity is not limited to the use of CleavaseTM enzyme, Taq DNA polymerase, E. coli DNA Various sources including limase I and eukaryotic structure-specific endonucleases (E.g., yeast RAD2 protein and RAD1 / RAD1). 0 complex [Harrington, J.J. And Liener (1994) Genes and Develop. 8: 1344] Mouse FEN-1 endonuclease (Harrington and Liener above) and offspring Bovine thymus 5 '→ 3' exonuclease [Murante, R.S. et al., (1994) J. Med. Biol. Chem. 269 : 1191]). In fact, this creates a unique cleavage product pattern for the substrate nucleic acid The actual experimental data is provided to show that a large number of enzymes can be used. CFLP^TM Enzymes shown here to be suitable for use in the method include Cleavase (Cleavase)^TMBN enzyme, Taq DNA polymerase, Tth DNA polymerase, Tfl Includes DNA polymerase, E. coli ExoIII and yeast RAD1 / RAD10 complex .   The present invention relates to an enzyme characterized in the literature to be a 3 'exonuclease Numerous enzymes, including CFLP^TMIndicates that it may be suitable for use in the method. The enzyme is CFLP^TM(Ie, substrate nuclei) To generate a unique cleavage product pattern for the acid) Is adopted. By carefully considering the steps described below, CFLP^TM Any enzyme ("enzyme X") can be evaluated for use in the method.   Initial CFLP^TMThe reaction is a previously characterized substrate nucleic acid [eg, human tyrosinase 157 nucleotide fragment of exon 4 of the gene (SEQ ID NO: 34). You. Substrate nucleic acid (about 100 fmol, this nucleic acid template allows for easy detection of cleavage products (Which may contain a 5'-end or other label) for the enzyme (ie, enzyme X) Solutions containing reaction conditions reported to be optimal for the characterized activity of Medium, put in thin-walled microcentrifuge tubes. For example, if the enzyme X is a DNA poly If it is a merase, the initial reaction conditions are optimal for the polymerization activity of the polymerase Will be used. If enzyme X is a polymerase If not, or if no specific component is reported to be necessary for activity If the initial reaction is a 1X CFLP with pH 7.2-8.2^TMBuffer solution (10 mM M OPS, 0.05% Tween-20, 0.05% Nonidet P-40), 0.2-1.0 mM MnCl_TwoContaining solution It can be assembled by putting it inside.   The substrate nucleic acid is denatured by heating the sample tube to 95 ° C for 5 seconds. Cool the reaction to a temperature suitable for the enzyme under test (eg, heat stable If limerase is being tested, the cleavage reaction will proceed at an elevated temperature, such as 72 ° C. Can do it. If the mesophilic enzyme is being tested, tube 37 for cleavage reaction. Cool to ° C). Following denaturation and cooling to the target temperature, the enzyme to be tested (ie, Enzyme X, if desired, 1X CFLP at pH 8.2^TMDilute in buffer The cleavage reaction is initiated by adding a solution containing 1-200 units of Let   Following the addition of the enzyme X solution, the cleavage reaction is allowed to proceed at the target temperature for 2-5 minutes. Next To terminate the cleavage reaction [this is a stop solution (95% formamide, 10 mM EDTA, 0.05% Bromophenol blue, 0.05% xylene cyanol) And the cleavage products are analyzed and detected using any suitable method (eg, Following electrophoresis on a denaturing polyacrylamide gel, transfer to a solid support and Isotope detection). CFLP for the generated cleavage pattern^TMBelow is the optimization test Check according to the criteria described.   Certain enzymes have a unique (ie, characteristic) cleavage product pattern from the substrate nucleic acid. CFLP if it can produce^TMSuitable for use in law. This cleavage depends on the presence of the enzyme Must be shown. In addition, the enzyme is activated by a given substrate under the same reaction conditions. It must be able to reproducibly generate the same cleavage pattern when split. Regarding reproducibility To test, the enzyme to be evaluated is manipulated on different occasions using the same reaction conditions. Used in at least two additional cleavage reactions. If both occasions are practically the same If a cleavage pattern is obtained, the enzyme can reproducibly generate the cleavage pattern, Hence CFLP^TMSuitable for use in law.   CFLP for enzymes derived from mesophilic bacteria^TMIf tested in reactions, they should first be 37 ° C Can be tested at However, these enzymes are used at relatively high temperatures in the cleavage reaction. May be desirable. Cleavage nucleic acid substrates over a range of temperatures The ability to detect sequence variations (ie, mutations) between different substrates whose cleavage reactions It is desirable when used for Strong secondary structure that can influence the cleavage pattern Is unlikely to be destabilized by a single base change, and therefore Naturally, it can interfere with mutation detection. So, using elevated temperature these solid structures Can be brought to the brink of instability, thereby effecting small changes in the array To be maximal and manifest as changes in the cleavage pattern it can. Mesophilic enzymes can be used at temperatures above 37 ° C under certain conditions known in the art. You. These conditions include high concentrations of glycemic (ie, 10-30%) in the reaction conditions. Includes the use of phosphorus. In addition, some enzymes can be isolated from mesophilic bacteria, Note that this fact alone does not imply that the enzyme may not be thermostable. Eyed. Therefore, CFLP^TMWhen testing the suitability of a mesophilic enzyme in a reaction, The reaction should be operated at 37 ° C. and higher. Or also the ratio Mild denaturants can be used to destabilize nucleic acid substrates at relatively low temperatures (E.g. 1-10% formamide, -10% DMSO and 1-10% glycerin are heat anxious Used in enzymatic reactions to mimic stabilization).   There are many types of nucleic acid substrates that can be analyzed using cleavage means such as 5 'nucleases. Both RNA and DNA are included. All such nucleic acid substrates are standard It can be obtained using progeny biological techniques. For example, the substrate is a tissue sample, tissue Can be isolated from cultured cells, bacteria or viruses and transcribed in vitro from DNA templates Or chemically synthesized. In addition, the substrate can be genomic material or plasmid. Or similar extrachromosomal DNA from bacteria, or Of such substances produced by treatment with restriction endonucleases or other cleavage agents. It can be a fragment or it can be synthetic.   Substrates can also be produced by amplification using PCR. Substrate is a single-stranded substrate If it should be a molecule, the substrate can be used for PCR with preferential amplification of one strand. Can be produced more (asymmetric PCR). Single-stranded substrates can also be conveniently produced by other methods. It can also be done. For example, two strands contain a biotin label at one end. A solid support (for example, a magnetic support) in which a double-stranded molecule having Gas beads). Biotin-labeled strand is streptor It is selectively captured by binding to the vidin-bead complex. Subsequent cleavage reaction It can be noted that can be performed using a substrate bound to a solid support. That Is the enzyme Cleavase^TMCan cleave the substrate that remains attached to the beads Because it can. A single-stranded substrate also exonucleates one strand from a double-stranded molecule. It can also be produced by digestion with creatase.   The nucleic acids in question may contain labels to aid their detection following the cleavage reaction. The label may be a radioisotope located at either the 5 'or 3' end of the nucleic acid (eg,³² P- or³⁵S-labeled nucleotide) or the label can be It can be distributed throughout the body (ie, an internally labeled substrate). The label is a non-isotopic detectable moiety such as a fluorophore that can be directly detected. Alternatively, it may be a reactive group that can be specifically recognized by a secondary reagent. For example, Biotinylated nucleic acid binds to an indicator (eg, alkaline phosphatase or fluorophore). Can be detected by searching using the combined streptavidin molecule, Or haptens such as digoxigenin are specific antibodies conjugated to similar indicators Can be detected. Alternatively, unlabeled nucleic acids are cleaved and stained (eg, By ethidium bromide staining or silver staining) or labeled probes Can be visualized by hybridization using In a preferred embodiment, the substrate Nucleic acids are labeled at the 5 'end with a biotin molecule and conjugated to alkaline phosphatase. Detection is performed using bound avidin or streptavidin. Another preferred In one embodiment, the substrate nucleic acid is labeled at the 5 'end with a fluorescein molecule and the anti- Detection is performed using a fluorescein antibody-alkaline phosphatase conjugate.   The cleavage pattern is essentially a partial digest of the substrate in the reaction. Substrate Labeling at the ends (eg, with biotin) ensures that all detectable fragments are Share the edge. Many of the structures recognized as active cleavage sites are only a few base pairs long There is a possibility, and Cleavase^TMRag used in the reaction It will be apparent that it is unstable at elevated temperatures. This in response to small sequence changes The formation or collapse of the structure causes a change in the cleavage pattern.   The product of the cleavage reaction is the assembly of fragments generated by the structure-specific cleavage of the input nucleic acid. Things. Analyze nucleic acids of different sizes and analyze them by electrophoresis, chromatography, Numerous, including fluorescence polarization, mass spectrometry and chip hybridization It can be analyzed by the method described above. The present invention will be described using electrophoretic separation. However It is noted that the analysis of cleavage products is not limited to electrophoresis. Electrophoresis book Selected to illustrate the method of the invention. Because electrophoresis is widely practiced in the industry And is easily accessible to the average practitioner. You.   If abundant amounts of DNA are available for analysis, direct detection of the cleavage The possibility of using light to analyze several samples in the same tube and the same gel It can be convenient to add. This “multiplexing” allows the wild-type and Automated comparison of closely related substrates such as mutants would be possible.   CFLP^TMReactions are useful for rapidly sorting out differences between similar nucleic acid molecules. Any A desired nucleic acid system (e.g., wild-type nucleic acid and one or more nucleic acids of wild-type nucleic acid). CFLP for natural mutant form)^TMFor optimal reaction, use the best CFLP^TMReaction strip Use one substrate from a test system (eg, a wild-type substrate) to determine Is most convenient. One preferred condition is that for other molecules of interest Comparison of CFLP^TMSelected to carry out the reaction. For example, cleavage reactions involve wild-type sequences. And then compare the mutant sequence to the wild-type pattern It can be cleaved under the same conditions. CFLP^TMThe purpose of optimization testing is to test molecules , A structure-specific cleavage agent such as Cleavase^TMEnzyme or DNAPT Treatment with aq is stable enough to give the signature sequence of the cleavage product, but the test molecule Small or single base changes in the DNA can cause significant changes in the sequence of cleavage products Will form a group (or group) of intrastrand structures that are sufficiently unstable Identification of such a condition set.   CFLP for use with single-stranded substrates as discussed below^TMExplain method optimization .   A panel of reaction conditions with variations in salt concentration and temperature was first performed to produce single-stranded CFLP.^TM The optimal set of conditions for "Optimal CFLP^TMIs an electrical It has the most uniform signal intensity between bands after electrophoretic separation, and Defined as a set of conditions that results in a set of occupied bands.   Two factors in the cleavage reaction that significantly affect the stability of nucleic acid constructs are that The temperature applied and the concentration of salt in the reaction solution. Similarly, formamide, urea Alternatively, other factors that affect nucleic acid structure, such as extreme pH, can be used. Initial testing typically involves three tests at four temperatures (50 ° C, 55 ° C, 60 ° C, and 65 ° C). Includes a total of 12 individual reactions performed at different salt concentrations (0 mM, 25 mM and 50 mM) Include. The present invention is not intended to be limited to the salts used. Use The salt used can be selected from potassium chloride, sodium chloride, etc., with potassium chloride being preferred. It is a good salt.   For each salt concentration to be tested, include DNA substrate, buffer and salt. Prepare 30 μl of the master mix. If the substrate is DNA, a suitable buffer Contains 3- [N-morpholino] propanesulfonic acid (MOPS) at pH 6.5-9.0, pH7 .2-8.4 is particularly preferred, and other `` good '' biological buffers, e.g., pH 6.5-9. Tris [hydroxymethyl] aminomethane (Tris) or N, N-bis [2- [Hydroxyethyl] glycine (Bicine), pH 7.5-8.4 is particularly preferred. Nuclear If the acid substrate is RNA, the pH of the buffer is reduced to the range 6.0-8.5, .0 is particularly preferred. If you plan to use manganese as a divalent cation in the reaction The use of Tris buffer is not preferred. If divalent cations are present in the buffer for a long time (eg For more than 5 minutes of incubation or storage of the original buffer). Manganese tends to precipitate as manganese (II) oxide in Tris There is. If manganese is to be used as the divalent cation, the preferred buffer is MO PS buffer.   For reactions without salt ("0 mM KCl" mixture), this mixture was 1.7 mM MnCl<sub>Two</sub>Or MgCl<sub>Two</sub>1X CFL containing (final concentration of divalent cation will be 1 mM) P<sup>TM</sup>Detect well for 5 digests in 30 μl buffer (10 mM MOPS, pH 7.2-8.2) (Eg, about 500 fmoles of 5 ′ biotinylated DNA or<sup>32</sup>P-5 'end End-labeled DNA about 100 fmol). Other concentrations of divalent cations may be Can be used if appropriate (eg, E. coli DNA polymerase I Usually 5mM MgCl<sub>Two</sub>Used in buffers containing). "25mM KCl" mixture is above In addition to the components, 41.5 mM KCl was included, and the “50 mM KCl” mixture was 83.3 mM KCl in addition to the above components. Includes Cl.   Mix the mixture in labeled reaction tubes (0.2 ml, 0.5 ml or 1.5 ml “eppend 6 μl each in a light mineral oil Or cover with a similar barrier and store on ice until use. MnCl<sub>Two</sub>Contains Not 1X CFLP<sup>TM</sup>60 μl containing the appropriate concentration of 5 ′ nuclease in buffer Assemble the enzyme dilution cocktail. Preferred 5 'nucleases and concentrations are cleaved Alease<sup>TM</sup>25-100ng of BN enzyme, 25ng or Taq DNA polymerase (Or other eubacterial Pol A DNA polymerase) preferable. MnCl<sub>Two</sub>Does not contain 1X CFLP<sup>TM</sup>Similar structure-specific cleavage in buffer Any suitable amount of tearing agent may be used.   If a strong (ie, stable) secondary structure is formed by the substrate, one Nucleotide changes significantly alter its structure or the cleavage pattern it produces. Not likely to change. Increased temperature to drive structures to the brink of instability Degree can be used, thereby maximizing the effect of small changes in the sequence and targeting Is manifested as a change in the cleavage pattern within the substrate, thus at that point A cleavage reaction can take place. As a result, the reaction was run at elevated temperatures (eg, That is, it is often desirable to operate above 50.degree.   Preferably, the reaction is performed at 50 ° C, 55 ° C, 60 ° C and 65 ° C. Each temperature to be tested As for the degree, three tubes at each of the three KCl concentrations were placed at 95 ° C. for 5 seconds. Keep and then cool to the selected temperature. Next, add 4 μl of enzyme cocktail to The reaction is started immediately. To assess nucleic acid stability under these reaction conditions Can contain three different tubes (enzymes in these tubes). Elemental or MnCl<sub>Two</sub>Does not contain 1X CFLP<sup>TM</sup>4 μl of buffer are poured). All reactions Proceeded for 5 minutes and 20 mM EDTA and 0.05% xylene cyanol and 0.05 Stopped by the addition of 8 μl of 95% formamide containing 5% bromophenol blue. To stop. The reactions are assembled on ice if necessary and stored. The completed reaction is Store on ice until all of the reaction series have been performed.   Heat the sample to 72 ° C. for 2 minutes and transfer 3-7 μl of each reaction to the appropriate gel For example, for nucleic acids up to about 1.5 kb, 7 M urine in 45 mM Tris borate buffer at pH 8.3 Element, 6-10% polyacrylamide containing 1.4 mM EDTA (19: 1 cross-linked), or Electrophoresis on natural or denaturing agarose gels for larger molecules Analysis. Nucleic acids are stained and autoradiographed (radio By isotope) or on nylon or other membrane support For subsequent hybridization and / or non-isotopic detection It can be more visualized. Inspect the generated pattern according to the criteria described above, And mutant comparison CFLP<sup>TM</sup>Reaction conditions are selected for the performance of.   "No enzyme" control assesses nucleic acid substrate stability under specific reaction conditions it can. In this case, the substrate is placed in a tube containing all reaction components except the enzyme. And perform the same treatment as the enzyme-containing reaction. Other control reactions can be performed. New Each time a mutant substrate is tested, the wild-type substrate can be cleaved. Or It has also been previously characterized in parallel with substrates suspected of containing different mutations. The mutants can also be engineered. By a previously characterized mutant Compares cleavage patterns generated by new test substrates with known cleavage patterns be able to. In this way, changes in the new test substrate can be identified.   CFLP generated by cleavage of single-stranded substrate<sup>TM</sup>If the pattern is overly powerful (ie If it contains a (strong) band, this indicates the presence of a very stable structure. Good Good signal redistribution modifies reaction conditions to increase structural stability (Eg, lower the cleavage reaction temperature; increase the monovalent salt concentration). This Other less stable structures can compete more effectively for cleavage.   CFLP<sup>TM</sup>If the reaction should be optimized for double-stranded substrate cleavage, take the following steps: To use. Cleavage of double-stranded DNA substrates up to 2,000 base pairs can be optimized in this way .   The double-stranded substrate may be labeled with a single end label using any method known in the art. It is prepared to contain. The molar amount of DNA used in the optimization reaction is single-stranded. It is the same as that used for optimizing a reaction utilizing a substrate. Single-stranded versus double-stranded groups Quality CFLP<sup>TM</sup>The most striking difference between the reaction optimizations is that the double-stranded substrate contains no buffer. MnCl in the reaction product<sub>Two</sub>The concentration is reduced to 0.2 mM, KCl (or other monovalent salt) is omitted and the enzyme concentration is 10-25n per reaction g. Variation in the concentration of monovalent salts (eg KCl) is critical Single-strand CFLP as a control factor<sup>TM</sup>In contrast to the reaction optimization (described above), double-stranded CF LP<sup>TM</sup>Temperature range is more critical for reaction optimization in reaction optimization Control factor. Double-stranded CFLP<sup>TM</sup>When optimizing the reaction, the substrate and The reaction tubes containing the other components described in (1) are at the following temperatures: 40 ° C, 45 ° C, 50 ° C, 55 ° C. C, 60 ° C, 65 ° C, 70 ° C, and 75 ° C You.   For each temperature to be tested, single-end labeled double-stranded DNA substrate and 15 μl volume Prepare a mixture containing an amount of distilled water and place in a thin-walled microcentrifuge tube. It is. This mixture can be coated with light mineral oil or liquid wax. (This coating is not generally required, but some Stranded DNA substrates will produce more consistent results).   MnCl<sub>Two</sub>Prepare a 2 mM solution of Each CFLP<sup>TM</sup>For the reaction, 10X CFLP<sup>TM</sup>Buffer 2 μl of 2 mM (100 mM MOPS of pH 7.2-8.2, 0.5% Tween-20, 0.5% Nonidet P-40)  MnCl<sub>Two</sub>2 μl and Cleavase<sup>TM</sup>Add 25ng of BN enzyme and distilled water Then, 5 μl of a diluted enzyme solution containing the final volume of 5 μl is prepared.   Heat the DNA mixture to 95 ° C for 10-30 seconds, then test each tube Reaction temperature (eg 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, and 75 ° C) Cool. For the cleavage reaction, add 5 μl of the diluted enzyme solution to each tube at the target reaction temperature. Start by doing Incubate the reaction at the target temperature for 5 minutes to terminate the reaction. (Eg, 95% formamide and 0.05% xyle containing 10 mM EDTA). Add 16 μl of stop solution consisting of cyanol and 0.05% bromophenol blue Addition).   The sample was heated to 72 ° C for 1-2 minutes and 3-7 μl of each reaction was added to For example, 7 M urine in 45 mM Tris borate buffer at pH 8.3 for nuclei up to about 1.5 kb Element, 6-10% polyacrylamide containing 1.4 mM EDTA (19: 1 cross-linked), or For electrophoresis on natural or denaturing agarose gels for larger molecules Analyze more. Nucleic acids are stained and autoradiographed (radio For oisotope) or nylon or other membrane support Transfer to the body, followed by hybridization and / or non-isotopic detection It can be visualized by output. Inspection of generated pattern according to the above criteria And double-stranded CFLP<sup>TM</sup>Reaction conditions are selected for the performance of. Control reactions are nucleic acid To assess stability or generate a reference pattern Can be operated.   Double-stranded CFLP<sup>TM</sup>When performing the reaction, use MnCl<sub>Two</sub>Preferably the concentration does not exceed 0.25 mM Would be good. If the end label on the double-stranded DNA substrate is lost (ie, When the product is detected, the 5′-end label is lost as judged by the disappearance of the signal). MnCl<sub>Two</sub>The concentration can be reduced to 0.1 mM. Any EDTA present in the DNA storage buffer Free Mn during reaction<sup>2-</sup>Will decrease the amount of Therefore, double-stranded DNA is It should be dissolved in Tris-HCl or water at a concentration of 0.1 mM or less.   A nucleic acid substrate may be attached at one end (eg, biotin or<sup>32</sup>Labeling with P) , All detectable fragments share a common end. Short DNA substrate (250 nu Enzymes (eg, Cleavase)<sup>TM</sup>BN) concentration And incubation length have minimal effect on signal intensity distribution However, this means that partial digestion of a single structure whose cleavage pattern is assumed by the nucleic acid substrate Rather than a relatively complete collection of stable structures formed by the substrate It indicates that it is digested. Relatively long DNA substrates (from 250 nucleotides) Large) have more chances of having multiple cleavage sites in each structure, and no residual full length The result is a clear overdigestion as indicated by the absence of the substance. these For DNA substrates, the enzyme concentration can be reduced in the cleavage reaction (eg, If initially 50ng of Cleavase<sup>TM</sup>Using BN enzyme and overdigestion Where apparent, enzyme concentrations can be reduced to 25, 10 or 1 ng per reaction. Wear). Alternatively, Mn to reduce the rate of cleavage<sup>2-</sup>And Mg<sup>2-</sup>Combination of CFLP<sup>TM</sup>Can be used in buffers. 0.2 mM MnCl as described above<sub>Two</sub>Is (one CFLP (with either single-stranded or double-stranded nucleic acid substrate)<sup>TM</sup>When used in reactions, Mn<sup>2-</sup> 1mM Mg in addition to<sup>2-</sup>The use of reduces the rate of cleavage. The 1059 bp DNA shown in FIG. In the case of the amplicon, the cleavage rate is reduced to about one third (Mn<sup>2-</sup>single Mn compared to Germany<sup>2-</sup>/ Mg<sup>2-</sup>In the mixture). 0.2-1.0mM Mn<sup>2-</sup>Substrate in the presence of If overdigestion is observed when incubating at the reaction temperature for 2-5 minutes , 0.2mM Mn<sup>2-</sup>/ 1mM Mg<sup>2-</sup>The mixture can be used with a reaction time of 5-20 minutes.   Generated by cleavage of either single-stranded or double-stranded substrates containing a biotin label The resulting cleavage product can be detected using the following non-isotopic detection method. below The description is provided for explanation only. One skilled in the art will have an alternative to detecting biotin-labeled products. I understand the alternative law. Separate the gel plate after electrophoresis of the reaction product And the gel remains flat on one plate. Cut to dimensions, 0.5X TBE (45mM Positively charged nylon pre-moistened in sborate, pH 8.3, 1.4 mM EDTA). Schleicher and Schuell, Keene, NH) on top of the exposed gel. gel Remove any air bubbles trapped between the membrane and By rolling a 10 ml pipette tightly across). Next, 3MM filter paper (Whatma n) Place the two pieces on top of the membrane, replace the other glass plate, and Tighten the sandwich with binder clips or press with a book or heavy object. This Progresses for 2 hours to overnight (signal increases with longer transition) ).   After transfer, carefully remove the membrane from the gel and air dry. Is it a squeezed bottle These distilled waters can be used to release the gel that has adhered to the membrane. Completely dry After that, the membrane was washed with 1.2X Sequenase Images blocking buffer (Se quenase Images Blocking Buffer (United States Biochemical, Cleveland, OH Remove any precipitate in the blocking buffer by decantation or filtration. Stir for 30 minutes. 1cm membrane<sup>Two</sup>0.3 ml of buffer is used per (Eg, 30 ml per 10 cm × 10 cm blot). Streptavidin-alkalipho Sphatase conjugate (SAAP, United States Biochemical) at 1: 4000 dilution Add directly to the blocking solution (avoid spotting directly on the membrane) And stir for 15 minutes. DH membrane<sub>Two</sub>Rinse easily with O, then membrane cm<sup>Two</sup>0.1% sodium dodecyl sulfate (SDS) using 0.5 ml buffer Wash three times in 1X SAAP buffer (100 mM Tris-HCl, pH 10, 5 mM NaCl) (1 Shake 5 minutes per wash). Rinse briefly with water between each wash. Next, 1mM membrane  MgCl<sub>Two</sub>Washes twice in 1X SAAP buffer (without SDS) containing Then put in a plastic heat sealable bag. Sterile pipette tip Use CDP-Star in the bag<sup>TM</sup>(Tropix, Bedford, MA) 0.05ml / cm<sup>Two</sup>Add the membrane Allow 5 minutes to distribute throughout the run. Remove any excess liquid and air bubbles from the membrane Remove, seal, and place membrane on X-ray film (eg, Kodak XRP) for 30 minutes. Expose for a minute. Exposure time is adjusted as needed for analysis and clarity You.   To date, CFLP<sup>TM</sup>All nucleic acid substrates tested in the system are reproducible fragments Generating a pattern. Due to the sensitivity and specificity of the cleavage reaction, Diagnosis of cancer, tissue classification, gene identification, bacterial and virus classification, polymorphism analysis, Mutation in genetic analysis, gene cross-over screening, etc. It is very suitable for rapid screening. This is augmented RNA analysis, high-level multiplexing and extension to longer fragments. Cleavase for nucleic acid characterization<sup>TM</sup>One clear use of reaction A significant advantage is that the pattern of cleavage products constitutes a characteristic fingerprint, Previously characterized mutants and ratios without sequencing possible mutants It can be compared. Also, the places where changes in the fragment pattern are observed suddenly change. Give a good index of different positions. But mutations need not be the exact site of cleavage Note that it only needs to be in an area that affects the stability of its structure Should. III. CFLP<sup>TM</sup>Of Mutations in the p53 Tumor Suppressor Gene Using the Assay   Tumor suppressor genes are important for tissue homeostasis Controls various other processes. One of the most widely studied of these, p5 The three genes not only can suppress the growth of cancer cells, but also (Levine, Annu. Rev. B) iochem. 62: 623 [1993]). Tumor suppressors alter or obliterate normal p53 function -Mutations are common.   Mutations in the p53 tumor suppressor gene are approximately half of all human cancers And it is the most common known change at the genetic level in the p53 gene Cancer-related genetic changes. The p53 gene is 3 Encodes a 53kD nuclear phosphoprotein consisting of 93 amino acids, which is cellular It is involved in the control of proliferation. Mutations in the p53 gene are generally (> 90% ), Amino acid equivalents rather than nonsense mutations that cause protein inactivation It is a missense mutation that causes a change in sex. High frequency of p5 found in human tumors 3 mutations indicate that missense mutations result in loss of tumor suppressor function and It is assumed to be due to the fact that it causes both gains of function (Lane, D .P. and Benchimol, S., Genes Dev. 4: 1 (1990)).   The gene encoding the p53 protein is large, spanning 20,000 base pairs, It is divided into 11 exons (see FIG. 4). Large p53 for the presence of a mutation The ability to scan genes has important clinical applications. In some major human cancers The presence of tumor p53 mutations is associated with poor prognosis. p53 mutation is node-negative Clinicians reach decision on more aggressive therapeutic treatment for reduced survival in breast cancer It has been shown to be an independent marker that is a finding that could help in the control. Also, Lowe and co-workers report that tumor cells are less susceptible to irradiation or chemotherapy. Significantly reduced by mutations that disrupt p53-dependent apoptosis (Lowe et al., Cell 74: 957 (1995)).   Regions of the p53 gene from approximately 10,000 tumors have been sequenced in the last 4-5 years Have been characterized, resulting in over 3,700 mutations, of which about 1,200 Are independent p53 mutations (ie, point mutations, insertions or deletions). De Database has been compiled and the European Molecular Biology Library (European Molecul ar Biology Laboratory (EMBL) data library and [Hollstein, M. et al., (1994) Nucleic Acids Res. 22: 3551 and And Cariello, N.F. et al. (1994) Nucleic Acids Res. 22: 3549]. In addition, the database IBM PC compatible software package developed to analyze information in [Cariello et al., Nucleic Acids Res. 22: 3551 (1994)]. Database A point mutation in the DNA was identified by DNA sequencing of the PCR amplification product. Most places Preliminary screening for mutations by SSCP or DGGE was performed.   Analysis of p53 mutations shows that the p53 gene is a highly conserved protein Close correlation between the main (ECD), specific to the transformation process Five Most Frequently Mutated Hotspot Regions of Human Tumors That Are Involved Area (HSR) is shown (see Figure 4. Bar height is associated with 5 HSRs) The relative percentage of total mutations shown is shown). Five HSRs are exon 5- 8 and is responsible for more than 85% of the detected mutations. However Et al., These studies generally indicate that their analysis is between PCR amplification and exons 5-8. Limited to sequencing of the region located, so fewer mutations outside this region are displayed You have to keep in mind what you have done. 10-15% of mutations Clinically effective p53 gene DNA diagnostics It must be able to scan for life-threatening mutations that have just been spread in a cost-effective manner.   The following table lists a number of known p53 mutations.   A. CFLP of p53 mutant in clinical samples<sup>TM</sup>analysis   To allow the identification of mutations in the p53 gene from clinical samples, A nucleic acid containing a gene sequence is prepared. Nucleic acid is genomic DNA form of p53 gene, RN It may include Form A or cDNA form. Nucleic acids are available from a variety of standard technologies. Or various clinical samples [new tissue or frozen Tissue, cell (eg, blood) suspension, cerebrospinal fluid, sputum, etc.]. example Qiagen offers kits that allow the isolation of RNA or DNA from tissue samples. . Inc. (Chatsworth, CA) and Stratagene (LaJolla, CA) respectively . Total RNA is isolated from tissues and tumors by several methods known to those skilled in the art Sa Qiagen. Inc. (Chatsworth, CA) provides protocols, reagents and plastic Tissue, cultured cells in about 20 minutes, providing the manufacturer with the manufacturer's instructions Alternatively, it allows for the isolation of total RNA from bacteria. For eukaryotic RNA isolates If it is desired to further concentrate the messenger RNA, It can be specifically isolated by binding to an oligo-deoxythymidine matrix. This Comparable isolation kits for both of these steps are available from several commercial suppliers. I can do it.   In addition, the RNA is conveniently 0.22 M NaCl, 0.75 mM MgCl<sub>Two</sub>, 0.1 M Tris-HCl, p H8.0, 12.5 mM EDTA, 0.25% NP40, 1% SDS, 0.5 mM DTT, 500 u / ml placental RNAase Homogenized tissue in buffer containing inhibitor and 200 μg / ml Proteinase K Upon lysis, it can be extracted from a sample containing the biopsy specimen. At 37 ° C After 30 minutes incubation, RNA is converted to phenol: chloroform (1: 1) And the RNA is recovered by ethanol precipitation.   Since most of the p53 mutants are found in exons 5-8, this region spans It is convenient to examine the PCR fragment to be used as the first analysis. Exons 5-8 Panning PCR fragments are amplified from clinical samples using RT-PCR (reverse transcription PCR) technology I can do it. A kit that allows users to generate PCR products starting from tissue is available from Perkin El-m er (Norwalk, CT) and Stratagene (LaJolla, CA). RT-PCR technology Selected segments of the coding region of a gene by using reverse transcription of RNA To generate a single-stranded cDNA corresponding to the Next, single-stranded cDNA is used as a template in PCR. Used. In the case of the p53 gene, an approximately 600 bp fragment spanning exons 5-8 The primers located in the coding region immediately adjacent to exons 5 and 8 by RT-PCR Produced using a marker. The PCR amplified segment is then subjected to a CFLP reaction, The reaction product is analyzed as described above in Section VIII.   Also, fragments suitable for CFLP analysis can be generated by PCR amplification of genomic DNA. D NA was extracted from the sample and corresponded to the sequences present in introns 4 and 8. Primer spans exon 5-8 containing intron 5-7 (about 2 kb fragment) Used to amplify a segment of the p53 gene. Updating DNA by CFLP reaction If it is desired to use smaller fragments for PCR may be selected to amplify small (<1 kb) segments, or large PCR A fragment may be split into two or more smaller fragments using restriction enzymes.   To facilitate identification of p53 mutants in clinical settings, first characterize A library containing CFLP patterns generated by the determined mutants is provided. You may. Patterns generated by cleavage of known and characterized p53 mutants Comparisons of patterns generated using nucleic acids from clinical samples can be found in patient tissues. Will allow for the rapid identification of a particular p53 mutant present in S. Libra Comparison of CFLP patterns from clinical samples to patterns present in This can be achieved by various means. The easiest and least expensive comparison involves a visual comparison U. Given the many unique mutants known at the p53 locus, That is, the manual) comparison is particularly useful when large numbers of clinical isolates are being screened. Might consume too much time. Therefore, the CFLP pattern or "Barco Mode may be prepared in electronic format for ease of comparison and efficiency. No. Electrical entries were made with reference p53 mutants (eg, GeneReader and Gel Doct or Fluorescence Geldocumentation system (BioRad.Hercules, CA) or the Im ageMaster (Pharma-cia Biotech. Piscataway. NJ)) It may include storage of a scan of the gel. In addition, the detection of the cleavage pattern is used for DNA sequencing. The banding pattern can be automated using the equipment (see Example 18). Can be stored as signals collected from appropriate channels during automated experiments. [Examples of suitable instruments for such analysis and data collection include fluorescence-based gels. An imager (imag-er) such as Molecular Dynamics and Hitachi Or real-time electrophoresis detection system, such as ABI 377 or Pharmacia ALF Fluoroimagers generated by DNA Sequencer].   B. Construction of a library of characterized p53 mutants   The creation of a library of characterized mutations makes clinical samples most popular. Can be screened rapidly and directly for the presence of Would. CFLP pattern generated from clinical samples against p53 barcode library Comparisons require costly and time-consuming DNA sequence analysis. Without establishing both the presence of the mutation in the p53 gene and its precise identification There will be.   The p53 barcode library is created using reverse genetics. p53 suddenly The engineering of the mutants was such that each engineered mutation was confirmed by DNA sequencing. Ascertain the identity and purity of each of the mutations. p53 barcodera Individual p53 mutants in the library were identified using a two-step “recombinant PCR” technique [Higuchi, R . (1991) In Ehrlich, H.A. (Ed.) PCR Technology: Principles and Applicat-ion s for DNA Amplification. Stockton Press. New York. pp.61-70 and Nel-son , R.M. and Long, G.L. (1989) Analytical Biochem. 180: 147] It is. FIG. 5 shows one of the two-step recombinant PCR techniques that can be used to generate p53 mutants. 1 shows a schematic diagram of the law.   The template for PCR amplification is the fully human p53 cDNA gene. First of two PCRs (Figure 5 , Referred to as "PCR1"), the oligonucleotide containing the engineered mutation (“Oligo A” in FIG. 5) and an oligonucleotide containing a 5 ′ arm of about 20 non-complementary bases Otide ("oligo B") amplifies a relatively small region (100-200 bp) of target DNA Used for The resulting amplification product contains a mutant at its extreme 5 'end, It will contain a foreign gene at the 3 'end. 3 'sequence is the directional clonin of the final amplified fragment (Important for sequencing and officially documenting DNA containing mutants) Designed to include a unique restriction site (eg, EcoRI). Upstream or first The product produced during one PCR is optionally used for this first PCR product during a second PCR. It may be gel purified before. However, in gel purification, this fragment was amplified by PCR. Once proved to be the only species done, it is not needed.   Next, a small PCR fragment containing the engineered mutant is the second round of PCR (PCR2) Used to guide In PCR2, the target DNA is the same subunit of p53 cDNA. Larger fragment (about 1 kb) of the loaned region. 3 'end arrangement of small PCR fragment Since the sequence is not complementary to any of the sequences present in the target DNA, mismatches are extremely Only that strand that occurs at the 5 'end is amplified by PCR2 (the 3' non-template arm is It cannot be extended by PCR). Amplification is a marker upstream of the engineered mutant locus. Of primer (“oligo C”) complementary to the target DNA region and PCR1 The addition of a primer ("oligo D") complementary to the 5 'non-complementary sequence of the product Is performed. This operation is sudden by inducing amplification from non-complementary sequences. Naturally, this results in specific amplification of only those sequences, including mutants. To a normal vector To facilitate cloning of these PCR products, a second unique restriction site is Go to C (eg, HindIII).   The use of this two-step PCR approach requires that each mutant be the initial set of Only one primer needs to be synthesized to be made after up ( That is, oligo A). oligo B, oligo C and oligo D all occur within a given area May be used. oligo C and oligo D contain different specific restriction sites So that these plasmids (eg, pUC19) Subsequent directional cloning of the PCR product is greatly simplified. Desired mutation Selective amplification of only those sequences containing changes in Simplify the configuration. What is the only way to confirm the sequence of the insert containing the plasmid Because it is needed. Once the insert sequence is confirmed, However, the clone containing the mutant may be maintained indefinitely as the bacterial master strain. In addition Thus, the DNA strain of each mutant can be maintained in the form of a large-scale PCR preparation. This It is included in a kit containing reagents for scanning for p53 mutants in clinical samples. As an individual control or as a supplemental master p53 mutant library control kit Harbors a plasmid containing the given mutant to be distributed as part of the Allows distribution of bacteria or PCR preparations.   Another two-step recombinant PCR is illustrated in FIG. 6 and described in Example 30. This one The method uses two mutagenic oligonucleotides (one for each strand). Are combined. These oligonucleotides are substantially complementary to each other , But with opposite orientations. That is, one is the upper strand or the sense strand. The "upstream" region of DNA with the mutant incorporated in the 3 'base region of the strand Are arranged to allow amplification of one strand, while the other strand is A "downstream" seg containing the intended mutation incorporated into the 5 'base region of the strand Arranged to allow amplification of the These two double-stranded products are They share the sequence provided by these mutagenic oligonucleotides. Spirit Anneal at the recessed 3 'end when made, bonded, denatured and annealed These strands are extended or filled in by the action of DNA polymerase. Thus, a full-length molecule having a mutant in the central region can be regenerated. This group The recombinant is the “outer” primer pair (the 5 ′ end of the “upstream” and the “downstream” intermediate Amplification can be achieved by the use of a primer pair used to create the 3 'end of the piece.   Since outer primers can amplify both recombinant and unmodified sequences, Another precaution needs to be taken in this way (compared to the method above), The method uses rapid recombination PCR to introduce foreign sequences using existing terminal primers. To be able to be done. In short, this method requires only few recombination. Often used when Large volume mutagenesis PCR should be performed. When this is the case, the first described method is preferred. What the first way is a single ol igo needs to be synthesized for each mutagenesis, This is because only the signal is amplified.   An important feature of kits designed for the identification of p53 mutants in clinical samples is the C Specific primers used to generate PCR fragments that should be analyzed for FLP Incorporation of DNA fragments from 100 bp to over 1500 bp are distributed by this technology. Analyze reproducibly and accurately for the presence of column polymorphisms, but with the same input The exact pattern arising from different length fragments of the DNA sequence will, of course, vary. U. The patterns are only shifted relative to each other according to the length of the input DNA Rather, in some cases, a greater extent of mutual interaction between distant regions of a long DNA fragment. Generate additional cleavage products not seen with short-acting input DNA products Can bring. For this reason, an exact match with the barcode library is The same as that used to generate the given version of the p53 barcode library Use primers designed to amplify fragments of the same size from the floor sample. Will be more secure.   C. CFLP specific for p53 mutant<sup>TM</sup>Pattern detection   CFLP<sup>TM</sup>The easiest and most direct way to analyze the DNA fragments generated in the reaction is It is based on gel electrophoresis. Electrophoresis is widely performed and readily available So the initial effort was to create a database in this well-known format Was intended to be. However, CFLP<sup>TM</sup>DNA generated by analysis It should be noted that elucidation of the fragments is not limited to electrophoretic methods. Massus Spectrometry, chromatography, fluorescence polarization, and chip hybridization All approaches that are currently being improved and developed in several laboratories It is. Once created, CFLP<sup>TM</sup>The database can be created using any of these methods. Even easily adopted for analysis.   There are several possible alternatives available for detecting CFLP patterns. Importance of CFLP analysis The benefit of a good user is that the result does not depend on the chosen method of DNA detection . DNA fragments are radioisotopes (eg, 5 'or 3' Ba<sup>32</sup>P or<sup>35</sup>S-labeled nucleotide), or the label (Ie, an internally labeled substrate). Label is non-isotopic detection Possible recognition, such as a fluorophore that can be directly detected, or a secondary agent. It may be a reactive group that enables it. CFLP pattern is immunostained, biotin-avi Detected by gin interaction, autoradiography and direct fluorescence imaging It has been. Radiation use is rapidly decreasing in clinical settings, Also, immunostaining and biotin-avidin based detection schemes can be applied to expensive membrane supports. Fluorescence-based detection methods are preferred because they require time-consuming migration of DNA. Maybe. However, none of the above methods can It is important to note that it can be used to create CFLP barcodes is there.   In addition to being a direct non-isotopic means of detecting CFLP patterns Fluorescence-based schemes offer additional notable advantages in clinical applications. CFLP allows analysis of several samples in the same tube and gel in the same lane . This "multiplexing" is achieved by individual analysis of each sample Fast and automated large numbers of samples in a fraction of the time at a lower cost than possible Allow comparisons. This approach opens the door to several other applications. the study One can double, triple or quadruple the number of samples to be tested on a given gel (up to four Dye in a single lane in a commercial DNA sequencer such as ABI 373/377 Have been proven to be available and compatible). Came. Also, analytes differ in pattern between the normal p53 gene and the putative mutant. of Differentiated labeling as internal standard to confirm both presence and correct location Through each tube and each gel lane with the size standards It may contain a normal p53 gene sample. VI. CFLP<sup>TM</sup>Detection and identification of pathogens using the method   A. Detection and identification of hepatitis C virus   Hepatitis C virus (HCV) infection is the leading non-A, non-B (NANB) hepatitis after blood transfusion worldwide. Cause. In addition, HCV is a major cause of hepatocellular carcinoma (HCC) and chronic liver disease worldwide. Is a major etiological agent. HCV infection is primarily transfusion recipient and intravenous drug use Transmitted to offspring, but maternal transmission to offspring and transmission to organ transplant recipients. It has been reported.   The genome of the positive strand hepatitis C virus contains 5 'and 3' non-coding regions. (Ie, the 5 'and 3' untranslated regions) and the core protein (C), The envelope glycoprotein (E1 and E2 / NS1) and the six nonstructural glycoproteins (NS2-N It contains several regions, including the polyprotein coding region encoding S5b). small Analysis of a large (9.4 kb) RNA genome has identified some regions of the genome. Very highly preserved during shedding, while other areas can change fairly quickly It was shown that. The 5 'non-coding region (NCR) is the most highly conserved region in HCV. You. These analyzes show that these viruses were divided into six basic genotype groups. And subsequently subdivided into 12 subtypes [of HCV genotypes Nomenclature and division have evolved: Altamirano et al. For a recent classification scheme. J. Inf-ect. Dis. 171: 1034 (1995)]. These virus groups Accurate identification of the substance during an outbreak is related to different geographic It is important to monitor the case. Only Group 1 HCV was observed in the United States, Numerous HCV genotypes have been observed in both Europe and Japan.   Also, the ability to determine the genotype of a virus isolate is sensitive to the different types of HCV. It allows comparison of clinical results from infections and infections by multiple types in a single individual. HCV is also associated with different efficacy of interferon treatment, Loop 1 infected individuals showed a slight response [Kanai et al., Lancet 39: 1543 (1992) And Yoshioka et al., Hepatology 16: 293 (1992)]. Infected individuals related to the virus type Place cleaning is more costly to the clinician and more costly, but It will make it possible to avoid fruitful drug treatments.   Existing methods for determining the genotype of HCV isolates include DNA sequencing or HCV-specific P of the segment of the HCV genome paired with hybridization to a sex probe CR amplification, RFLP analysis of PCR amplified HCV DNA, and the like. Of these methods All suffer from the limitations described above (ie, DNA sequencing is too labor intensive). Intensive and expensive, not practical for clinical study setting; RFLP analysis Has a problem with low sensitivity).   Universal and genotype-specific primers extracted from plasma or serum RNA [Okamoto et al., J. Gen. Virol. 73:67 3 (1992); Yoshioka et al., Hepatology 16: 293 (1992) and Altamirano et al., Supra. ]. These primers are the CFLP of the present invention.<sup>TM</sup>Can be used as a substrate in assays Can be used to generate a different PCR product. As shown herein, CFLP<sup>TM</sup>analysis Provides a rapid and accurate method of typing HCP isolates. CFLP for HCV substrate<sup>TM</sup>Minute The analysis makes it possible to distinguish between the major genotypes and subtypes of HCV, Thus, there is provided an improved method of genotyping HCV isolates.   B. Detection and identification of multidrug-resistant Mycobacterium tuberculosis   Over the past decade, there has been a tremendous resurgence in the incidence of M. tuberculosis in this country and around the world. Was. In the United States, the incidence of Mycobacterium tuberculosis has risen steadily over the past decade, with 2,000 deaths annually The disease caused as many as 10 million Americans to become infected. The situation is It is significant in New York City, where the incidence has more than doubled in the last decade, with 19 In 1990, this represented 14% of all new cases in the United States [Frieden et al., New Eng. l.J.Med. 328: 521 (1993)].   The crisis in New York City is particularly disastrous. What is going on This is because a proportion (about 1/3) is resistant to one or more antituberculosis drugs [F-rieden And Hughes, Scrip Magazine May (1994)]. Mycobacterium tuberculosis (M (DR-TB) is an iatrogenic disease caused by incomplete treatment of primary infections [Jac-obs, Jr. , Clin. Infect. Dis. 19: 1 (1994)]. MDR-TB is a particularly serious squirrel in immunocompromised people It is clear that these individuals are likely to be other healthy individuals. Infected with MDR-TB strain [Jacobs, Jr., supra]. Mortality of immunocompromised individuals Is surprisingly high, compared to a mortality rate of less than 50% in other unimpaired individuals. % Often [Donnabella et al., Am. J. Respir. Dis. 11: 639 (1994)].   From a clinical point of view, M. tuberculosis has a very long onset time and other Always difficult to diagnose due to environmental morbidity of fast growing Mycobacterium species . The doubling time of M. tuberculosis is 20-24 hours, and growth by the usual method is It typically takes 4-6 weeks to identify a bacterium as positive [Jacobs, Jr. et al., Science. 260: 819 (1993) and Shinnick and Jones Tuberculosis: Pathogenesis. Prote ct-ion and Control. Bloom. Ed., American Society of Microbiology. Washing ton, D.C. (1994), pp. 517-530]. Further to diagnose drug sensitivity of a given strain It can take 3-6 weeks [Shinnick and Jones, supra]. Needless to say Patients may or may not be symptomatic, but almost certainly What are the health risks to infected individuals and the public during the delay period that are It's a thing. Once the drug resistance profile has been elucidated and diagnosed, Treatment of a single patient can cost up to $ 250.000 and can take up to 24 months.   The tragic risks imposed by MDR strains and the recent surge in the incidence of the disease In combination, the detection of M. tuberculosis and the drug resistance of M. tuberculosis clinical isolates Research activities aimed at accelerating the elucidation of the feel Promoted the explosion of industrial development. Some of these methods require that certain strains be Mainly for the purpose of measuring mycobacterium species other than mycobacterium or mycobacterium Be concentrated. Both culture-based and nucleic acid-based methods have been developed, Method allows for more rapid identification of M. tuberculosis than the classical method. Make it work. Detection time from 6 weeks or more to only 2 weeks (culture-based method) or 2 days (Method based on nucleic acid). Culture-based methods are now being spread to clinical laboratories. Some of the rapid nucleic acid-based methods that have been used, but can be applied directly to clinical samples. A few are under development. Non-Mycobacterium spp. Clinical samples such as sputum need to be first "decontaminated" to reduce contamination (Usually performed by pretreatment with N-acetyl L-cysteine and NaOH). Shinnick and Jones, supra).   The polymerase chain reaction (PCR) has been applied for the detection of M. tuberculosis, It can be used to detect its presence directly from clinical specimens within days. Good sensitivity New technologies rely on two-step operation. The first step is the PCR amplification itself and the second step Is a hybrid of amplicons to M. tuberculosis-specific oligonucleotide probes Analysis or analysis by RFLP or DNA sequencing [Shinnick and Jones, supra.].   Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe) Transcription Mediated Amplication [TMA; to amplify target rRNA sequence Essentially self-sustained sequence reaction (3SR) amplification]. rRNA sequence is amplified once And then it is detected by a dye-labeled assay such as PACE2. This assay Is highly inhibited by substances present in clinical samples.   Cycling Probe Reaction (CPR; ID Biomedical). Detect the presence of Mycobacterium tuberculosis This technology, which is being developed as a diagnostic tool for Set. For signal amplification, a three-layer DNA-RNA-DNA probe can be used, such as a M. tuberculosis-specific sequence. By forming a hybrid with the target nucleic acid. After adding RNAse H The RNA portion of the chimeric probe is degraded, releasing the DNA portion, which Shows that the target sequence is present [Yule. Bio / Technology 12: 1 335 (1994)]. Need to use RNA probes, especially for use in crude clinical samples This is a disadvantage, in which case RNase contamination is often severe.   The nucleic acid-based detection and discrimination methods are more evident than conventional culture-based methods Give you a great time saver. Although they are beginning to enter clinical settings, human tuberculosis Their usefulness in routine diagnosis of fungi has largely shifted to culture-based methods. It remains a problem because of the problems associated with differential contamination and low sensitivity. in addition, Many of these procedures are restricted to the analysis of respiratory samples [Yule. Bio / Technology 12 : 1335 (1994)].   ii) Determination of antibiotic resistance profile of M. tuberculosis       a) Culture-based method: Once the positive identification of M. tuberculosis has been made, It is necessary to characterize the degree and nature of the resistance of the strain to it. Antibiotics The conventional method used to measure resistance is the proportional agar dilution method, The culture diluent is applied to the medium containing antibiotics and the control medium without antibiotics. Clothed. This method is typically required for the diagnosis and characterization of unknown clinical samples. Add an additional 2-6 weeks to the required time [Jacobs, Jr. et al., Supra].   Luciferase reporter Mycobacteriophage (LRM) assay 199 First described in 3 years [Jacobs, Jr. et al., Science 260: 819 (1993)]. This asse In b, a mycobacterial phasor containing a cloned copy of the luciferase gene Used to infect mycobacterial cultures. Luciferase Expressed luciferase in the presence of ATP and ATP Produces photons that are easily distinguishable, and is responsible for the growth of Mycobacterium in the presence of antibiotics. Enables accurate measurement of degree. Once a sufficient culture is obtained (usually after inoculation 10-14 days), the assay can be completed in 2 days. This method is specific to LRM for M. tuberculosis. There is a problem with the fact that it is not unusual. They are also Mycobacterium smeg M. smegmatis and Mycobacterium bovis (e.g., BCG ), Thereby complicating the decoding of positive results. Discrimination between the two species Is due to growth in a special medium that does not support the growth of M. tuberculosis (eg, NAP medium). Needs to be done. This confirmation takes another two to four days.   The above culture-based method for measuring antibiotic resistance is a putative novel anti-mycotic Assess the efficacy of bacterial agents and drugs for which genetic targets have not yet been identified Will continue to play a role. However, it includes many of the frontline drugs. Elucidate the molecular basis for resistance to several anti-mycobacterial agents Recent success has been based on extremely fast, more accurate and more informative DNA polymorphisms. Enabled use of the assay.   b) DNA based method: isoniazid, rifampin, streptomycin, The loci associated with resistance to fluoroquinolones and ationamides are the same. Jacobs, Jr. et al., Supra; Heym et al., Lancet 344: 293 (1994) and And Morris et al., J. Infect. Dis. 171: 954 (1995)]. Pyrazinamide and ethambu Isoniazid (inh) and rifamppi with toll or streptomycin Rif combination is the first line of attack against confirmed cases of M. tuberculosis Used routinely [Banerjee et al., Science 263: 227 (1994)]. Therefore, Resistance to one or more of these drugs is disastrous for short course chemotherapy treatment Can have various meanings. The increasing incidence of such resistant strains has detected them, The cost of invalid and possibly harmful treatments that continue thereby and the social health Requires the development of rapid assays that reduce the risk of Genetics associated with drug resistance Rapid Screening of Nucleotide Changes That Identify Some Locus Can Lead to Drug Resistance And promoted the use of mutation detection techniques for signaling. Large numbers of targets directly from clinical specimens Of amplification procedures, such as PCR and SDA, that have successfully replicated specific DNA Is a DNA-based approach to antibiotic profiling based on normal culture To be much quicker than the method.   The most widely used technique for gene identification of mutants conferring drug resistance is DN A sequencing, Restriction Fragment Length Polymorphism (RFLP), PCR-Single   Stranded Conformational Polymorphism (PCR-SSCP) and PCR-dideoxyfin g-erprinting (PCR-ddf). All of these technologies have the shortcomings described above. Have a point. Each of them accurately and specifically identifies individual alleles Does not provide a quick, reproducible means.   In contrast, the CFLP of the present invention<sup>TM</sup>The method is structured to produce a distinct recovery of DNA fragments. Provides an approach that relies on specific cleavage. This method detects sequence polymorphisms. Highly sensitive in ability (> 98%), time, skill and cost of the above technology Need some of.   CFLP to MDR-TB detection<sup>TM</sup>Application of method amplified from rpoB and katG genes Described herein using segments of DNA that have been described. Isoniazid (inh A), streptomycin (rpsL and rrs), and fluoroquinoline (gyrA) MDR-TB, including but not limited to genes involved in conferring resistance Other genes associated with CFLP<sup>TM</sup>Equally well suited for assays.   C. Detection and identification of bacterial pathogens   Identification and typing of bacterial pathogens are important for clinical management of infectious diseases. Microorganism Accurate identification of antibiotics is not only used to distinguish symptoms from health On the basis of deciding which quality or other antimicrobial therapy is best for treatment is there. Traditional methods of pathogen typing include growth characteristics, color, cell or colony With specific antibodies to identify morphology, antibiotic susceptibility, staining, odor and bacteria Various phenotypic features were used, including reactivity. All of these methods are suspicious Requires the cultivation of pathogens, which results in high material and labor costs, Rugged, false positives due to mishandling and small numbers of viable cells or many pathogens There are several significant drawbacks, including false negatives due to sensitive culture requirements. doing. In addition, culture methods require a relatively long time to obtain a diagnosis, Due to the potentially life-threatening nature of such infections, Antibiotic therapy is often started. In many cases, the pathogen has a normal flora It is very similar to its constituent organisms and can be distinguished from harmless strains by the methods cited above. May not come. In these cases, the determination of the presence of pathogenic strains has recently been developed. It may require a high degree of elucidation provided by advanced molecular typing methods.   Several test methods for genetic material from the organism concerned have been developed. Of this type One way to perform the analysis is to use species-specific DNA or RNA from the organism to be tested. By hybridization of a target nucleic acid probe. This should be tested Immobilize denatured nucleic acids on a membrane support and only in the presence of DNA or RNA from pathogens This can be done by probing with bound labeled nucleic acids. In this way, the pathogen Can be determined. Organisms can be further distinguished using the RFLP method described above, in which case Genomic DNA is electrophoretically separated and supported on nitrocellulose or nylon membrane Before being transferred to the body, it is digested with one or more restriction enzymes. For species-specific nucleic acid probes Exploration is reproducible, identifying strains if they indicate a change between isolates It will reveal a banding pattern that can be used as a method. But However, these methods have the shortcomings outlined above. Hybridization -Based assays are time consuming and require less stringency of hybridization. Inaccurate or misleading results if the sea is not well controlled There RFLP identification also depends on the presence of suitable restriction sites in the DNA to be analyzed.   To address these concerns regarding hybridization and RFLP as diagnostic tools Some approaches to molecular analysis based on polymerase chain reaction (PCR) amplification to address Law had gained popularity. One good, called PCR fingerprinting In a well-accepted method, the size of the fragment generated by PCR is used as an identification tool. Used. In this type of assay, the primers have a variable number of tandem repeats (true In nuclear organisms, it is targeted to the region containing the VNTR). Number of repeats, and thus The length of the PCR amplicon is characteristic of a given pathogen, and these Co-amplification of several loci produces specific and reproducible fingerprints Thus, it is possible to enable discrimination between closely related species.   However, in some cases where organisms are closely related, the target of amplification is size In order to obtain a more accurate identification without showing any differences in Needs to be explored. This is similar to whole genome hybridization described above. May be performed on a solid support in a manner similar to that of the assay. Have a gender problem. In addition, the inside of the PCR fragment contains sequence-specific binding events. Can be used as a template. As outlined above for LCR, this method Single-stranded probes to be matched are arranged along the relevant sequence on both sides of the identified polymorphism, As a result, the success or failure of binding is determined by the specific nucleotide sequence at that site. Will indicate presence or absence. Hybridization method for PCR product analysis Or the binding method provides advance knowledge of the exact sequence in the region of probe binding. And differences outside the probe binding region are not detected. These methods Is less suitable for testing and typing of poorly characterized new isolates I haven't.   In the method of the present invention, a plasmid recognizing a conserved region of a bacterial ribosomal RNA gene is used. Imers allow amplification of the segments of these genes that contain the site of change. Ribosomal gene sequence changes distinguish between similar organisms at the DNA sequence level Not only has it become an accepted method, but their consistent rate of change It allows the sequence to be used to assess the evolutionary relationships of an organism. That is, The more similar the nucleic acids are at the sequence level, the closer the organism under consideration is considered to be [Woese, Bacterial Evolution. Microbiological Reviews, vol 51, No. 2, 198 7]. The present invention relates to the amplification products derived from these sequences as individual barcodes (ie, , Cleavage patterns) can be used to generate highly Also allows for the detection of sequence polymorphisms without prior knowledge of the existence of said polymorphism. You. With proper choice of primers, all amplifications are comprehensive (eg, Ratio of the distantly related organisms (using the stored ribosome sequence). Allows the primers to be compared or is very specific for a given genus Can be selected to allow testing at the species and subspecies level. Trial of ribosomal gene Experiments are extremely useful for these characterizations, but CFLP in bacterial typing<sup>TM</sup>One The use of the method is not limited to these genes. Certain growth characteristics (eg, carbon source selection) Selection, antibiotic resistance, methicillin resistance or antigen production), or special Includes, but is not limited to, genes associated with vesicle morphology (eg, pili formation) , Other genes are CFLP<sup>TM</sup>Equally well suited for assays.   D. Extraction of nucleic acids from clinical samples   CFLP<sup>TM</sup>Use assays to detect and identify microorganisms in clinical samples. Nucleic acids are extracted from a sample to provide a nucleic acid substrate for. Nucleic acids are species Various clinical samples [fresh sets] using various conventional techniques or commercially available kits Tissue or frozen tissue, cell (eg, blood) suspension, cerebrospinal fluid, sputum, urine, etc.] Can be extracted. For example, it allows the isolation of RNA or DNA from tissue samples. Kits from Qiagen. Inc. (Chatsworth, CA) and Stratagene (LaJolla, CA). I can do it. For example, the QIAamp Blood kit contains blood (fresh, frozen or dried) and Allows isolation of DNA from bone marrow, body fluids or cell suspensions. QIAamp organization kit Allows the isolation of DNA from tissues such as muscle, organs and tumors.   A relatively uniform specimen (eg, blood, bacterial colonies, viral plaques, or Cerebrospinal fluid) from more complex specimens (eg, urine, sputum or feces) Are also well suited for use as templates for amplification of specific PCR products. [Shibata PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkh- auser, Boston (1994), pp. 47-54]. Of the substance to be amplified (ie, the target nucleic acid) Samples containing relatively few copies, such as cerebrospinal fluid, can be added directly to PCR . Blood samples had a special problem with PCR due to the inhibitory nature of red blood cells. Red blood The spheres need to be removed before using the blood during PCR. For this purpose, classic There are both standard and commercially available methods [eg, QIAamp Blood Kit , Passing through a Chelex 100 column (BioRad), etc.]. Target chosen for direct detection of M. tuberculosis Extraction of nucleic acid from sputum, a book, kills other bacterial species before decontamination, or It needs to suppress its growth. This decontamination typically involves N-acetyl L- This is done by treating the sample with cysteine and NaOH (Shinnick and J ones, supra). This decontamination process occurs when sputum specimens are cultured before analysis. Only necessary.   Experiment   The following examples serve to illustrate certain preferred embodiments and aspects of the present invention. Yes, and should not be considered as limiting the scope of the invention.   In the following disclosure, the following abbreviations apply: ° C. (Celsius); g (Gravity 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); IVS (intervening sequence); p (plasmid); μl (microliter (Ml); ml (milliliter); μg (microgram); pmol (picomolar); mg (milligram); M OPS (3- [N-morpholino] propanesulfonic acid); M (mole / liter); Mol / liter); μM (micromol / liter); nm (nanometer); kdal (kilo Luton); OD (optical density); EDTA (ethylenediaminetetraacetic acid); FITC (fluorescein) Isothiocyanate); SDS (sodium dodecyl sulfate); NaPO<sub>Four</sub>(Sodium phosphate ); Tris (tris (hydroxymethyl) aminomethane); PMSF (phenylmethylsulfo TBE (tris-borate-EDTA, i.e., with boric acid rather than HCl) Tris buffer containing EDTA); PBS (phosphate buffered saline); PPBS (1 mM PMSF Phosphate buffered saline); PAGE (polyacrylamide gel electrophoresis); Tween Loxyethylene-sorbitan); Boehringer Mannheim (Boehringer Mannheim, I ndianolis, IN); Dynal (Dynal A.S., Oslo, Norway); Epicentre (Epicentre Technologies, Ma-dison, WI); National Biosciences (Na National Biosciences, Plymouth, MN); New England Biolabs (New England Biolab) s, Beverly, MA); Novagen (Novagen, Inc., Madison, WI); Perkin Elmer (Perkin  Elmer, Norwalk, CT); Promega Corp. (Promega Corp., Madison, WI); RJ Res earch (RJ Research, Inc., Wat-ertown, MA); Stratagene (Stratagene Cloning S ystems, La Jolla, CA); USB (U.S. Biochemical, Cleveland, OH).   Example 1   Properties of natural thermostable DNA polymerase   A. 5 'nuclease activity of DNAPTaq   Polymerase chain reaction (PCR) [Saiki et al., Science 239: 487 (1988); Mullis and And Faloona, Methods in Enzymology 155: 335 (1987)], DNAPTaq Not many DNA sequences can be amplified. Can be amplified using DNAPTaq One sequence is shown in Figure 7 (the hairpin structure is SEQ ID NO: 15 and the primer is Are SEQ ID NOs: 16 to 17). This DNA sequence folds itself into two Hairpipes with main-chain arms (these correspond to the primers used for PCR) Has the remarkable characteristic that it can form   Test if this failure of amplification is due to the 5 'nuclease activity of the enzyme To achieve this, we used DNAPTaq to amplify this DNA sequence during 30 cycles of PCR. And the performance of DNAPStf were compared. Wisconsin-Madison Obtained from the Biotechnology Center at the University of New York. DNAPTaq and DNAPStf to Pe rkin Elmer (ie, AmpliTaq DNA polymerase and Ampli-Ta q Stoffel fragment of DNA polymerase). Substrate DNA is cloned into pUC19 in double-stranded form. The hairpin structure shown in FIG. Primers used for amplification As SEQ ID NOs: 16-17. Primer SEQ ID NO: 17 is a hairpin in FIG. Shown annealed to the 3 'arm of the structure. Primer SEQ ID NO: 16 is in FIG. Shown as the first 20 nucleotides of the bold of the 5 'arm of the hairpin .   Polymerase chain reaction solution is supercoiled in 50 μl of 10 mM Tris Cl pH 8.3. 1 ng of plasmid target DNA, 5 pmole of each primer, each dNTP4 It contained 0 μM, and 2.5 units of DNAPTaq or DNAPStf. DNAPTaq reaction solution Contains 50 mM KCl and 1.5 mM MgCl<sub>Two</sub>Was included. Temperature profile during 30 cycles At 95 ° C for 30 seconds, 55 ° C for 1 minute and 72 ° C for 1 minute. 10 of each reaction % Of polyammonium in a buffer of 45 mM Tris Borate, pH 8.3, 1.4 mM EDTA (0.5X TBE). Analyzed by gel electrophoresis in rilamide (crosslinked 29: 1).   FIG. 8 shows the results. The expected product is DNAP instead of DNAPTaq (shown as `` T '') Made by Stf (shown simply as "S"). The present inventors have determined that the DNAPTaq It is concluded that rease activity is responsible for the lack of amplification of this DNA sequence.   5 ′ unpaired nucleotides in the substrate region of this structural DNA are removed by DNAPTaq. 4 cycles of PCR using the same polymerase to test The fate of the middle end-labeled 5 'arm was compared (Figure 9). DNAPStf and³²P-5 'terminal mark A hairpin mold such as the hairpin mold described in FIG. I made it. The 5 'end of the DNA was cut by DNAPTaq rather than DNAPStf by several large Released as strips. The size of these fragments (based on their motility) Contains most or all of the unpaired 5 'arm of DNA. Thus, cleavage Occurs at or near the base of the branched duplex. These released fragments are directly distributed. Sequence analysis and extended by terminal deoxynucleotidyl transferase Terminate at the 3'OH group as evidenced by the ability of the fragment to   Figure 10-12 was designed to characterize the cleavage reaction catalyzed by DNAPTaq The results of the experiment are shown. Unless otherwise noted, cleavage reactions were 10 mM in a total volume of 10 μl. Tris-Cl, pH 8.5, 50 mM KCl and 1.5 mM MgCl_Two0.01 pmole heat-denatured end label Hairpin DNA (there are also unlabeled complementary strands), 1 pmole primer (3 'Complementary to the arm) and 0.5 units of DNAPTaq (estimated to be 0.026 pmole). I was out. As shown, some reactions have different concentrations of KCl, and The exact times and temperatures used for these experiments are shown in the individual figures. Includes primer The primer shown in FIG. 6 was used as the reaction solution (SEQ ID NO: 17). In some cases Is prepared by providing a polymerase and selected nucleotides. The immer was extended to the binding site.   Reaction MgCl_TwoOr started at the final reaction temperature by addition of enzyme. Reaction 20 mM ED 8 μl of 95% formaldehyde (stop solution) containing TA and 0.05% marker dye The addition was stopped at their incubation temperature. National Bioscien-ce Oligo from s, Inc.^TMUsing primer analysis software, the listed T_m A calculation was made. Using 0.25 μM as DNA concentration with 15 or 65 mM total salt, These were measured (1.5 mM MgCl in all reactions)._TwoCalculated the value of 15 mM salt for these calculations. Given the).   FIG. 10 shows an autoradiograph containing the results of a set of experiments and conditions for the cleavage site. It is. FIG. 10A is a measurement of a reaction component that allows for cleavage. 5 'end labeled hairpi Incubation of DNA was performed at 55 ° C. for 30 minutes using the indicated components. . The product is resolved by denaturing polyacrylamide gel electrophoresis and the product length ( (Unit: number of nucleotides). FIG. 10B shows the cleavage site in the absence of the added primer. Describe the effect of temperature on The reaction is allowed to proceed for 10 minutes at the indicated temperature in the absence of KCl Incubated. Indicates the length (unit: number of nucleotides) of the product.   Surprisingly, DNAPTaq cleavage requires either primers or dNTPs. No (see FIG. 10A). Thus, 5 'nuclease activity is related to polymerization I can't. Nuclease activity requires magnesium ions, but magnesium Mions can be substituted with potential changes in specificity and activity. Zinc ion Or none of the calcium ions support the cleavage reaction. Reaction is from 25 ° C to 85 ° C And the rate of cleavage increases at higher temperatures.   Referring to FIG. 10, primers are not extended in the absence of added dNTPs. However Thus, primers affect both the site and rate of hairpin cleavage. Cleavage part The change in position (Figure 10A) is due to the break in the short duplex formed between the arms of the DNA substrate. It is clear that this occurs. In the absence of primer, the underlining in FIG. The more shown sequences could pair to form an extended duplex. Extension Cleavage at the end of the resulting duplex was observed in lane 10A without the addition of primer. Will release the 11 nucleotide fragment seen. Addition of excess primer (Figure 10A, lanes 3 and 4) or high temperature incubation (FIG. 10B) Disrupts the short extension of the heavy chain, resulting in a long 5 'arm and thus a long cleavage product.   The position of the 3 'end of the primer can affect the exact site of cleavage. Electrophoresis analysis In the absence of a primer, the cleavage occurs between the first and second base pair substrate duplex (warmth). (In extended or shortened form, depending on the degree). Step When the primer extends to the base of the duplex, cleavage also occurs at one nucleoside in the duplex. This produces tides. However, a primer of 4 or 6 nucleotides gap The cleavage site is 4 nucleotides in the 5 'direction when present between the 3' end of 6 nucleotides.   Figure 11 shows the presence (Figure 11A) or absence (Figure 11B) of the primer oligonucleotide. The kinetics of cleavage is described. Reaction with 50 mM KCl (Figure 11A) or 20 mM KCl (Figure 11B) At 55 ° C. Decompose reaction products by denaturing polyacrylamide gel electrophoresis And the length of the product (unit: number of nucleotides). "M" for marker is 5 ' End-labeled 19-nt oligonucleotide. Under these salt conditions, FIGS. B indicates that the reaction is about 20 times faster in the presence of primer than in the absence of primer. Indicates that it is clear. This effect on efficiency is due to the proper sequence of the enzyme on the substrate. And contribute to stabilization.   When both reactions are performed in 50 mM KCl, the relative shadow of the primer on the cleavage rate The sound becomes extremely large. In the presence of primer, the rate of cleavage is up to about 50 mM in KCl concentration. Increase with degree. However, suppression of this reaction in the presence of primer It is evident at 0 mM and complete at 150 mM KCl. In contrast, in the absence of primer Is increased by the concentration of KCl up to 20 mM, but at concentrations above 30 mM It is lowered by. At 50 mM KCl, the reaction is almost completely suppressed. Absence of primer The inhibition of cleavage by KCl below is affected by temperature and is even more pronounced at lower temperatures.   Recognition of the 5 'end of the arm to be cleaved appears to be an important feature of substrate recognition It is. Substrates lacking a free 5 'end, e.g., circular M13 DNA, It cannot be cleaved under the conditions of Even 5′-armed substrates can be opened by DNAPTaq. The speed of the crack is affected by the length of the arm. In the presence of primer and 50 mM KCl The cleavage of the 5 'extension, which is 27 nucleotides in length, is substantially complete within 2 minutes at 55 ° C I do. In contrast, fractions with 5 'arms of 84 and 188 nucleotides. The cleavage of the offspring is only about 90% and 40% complete after 20 minutes. B. Incubation reduces the effect of suppressing long elongation indicating secondary structure in the 5 'arm, Alternatively, a thermolabile structure in the enzyme may inhibit the reaction. Run under conditions of excess substrate The mixing experiments described above show that molecules with long arms are non-productive complexes (non-productive complexes). complex)). These results That the 5 'nuclease domain moves the 5' arm from one end to the other May indicate gaining access to the cleavage site at the end of the branched duplex. Longer 5 'arms are expected to have more accidental secondary structure (especially KCl concentration This is probably preventing this movement.   Cleavage should be at least 2 kilobases substrate strand target molecule or pilot It is clear that it is not suppressed by the long 3 'arm of the nucleic acid. In other extremes The 3 'arm of a pilot nucleic acid as short as one nucleotide is not efficient Can support cleavage in a primer independent reaction. Well matched pair Ligonucleotides do not trigger cleavage of the DNA template during primer extension.   Open the molecule even when the complementary strand contains only one unpaired 3 'nucleotide. The ability of DNAPTaq to cleave may be beneficial in optimizing allele-specific PCR Not. PCR primers with unpaired 3 'ends are pilot oligonucleotides Potential with DNAPTaq in the absence of nucleoside triphosphates acting as Of unwanted templates during pre-incubation of the active template-primer complex Alternative cleavage could be induced.   B. 5 'nuclease activity of other DNAPs   Determine if other 5 'nucleases in other DNAPs are suitable for the present invention To do this, an array of enzymes (some of which have 5 'nuclease activity apparent in the literature) (Reported to be without). Cleave nucleic acids in a structure-specific manner Reports the performance of these other enzymes as being optimal for synthesis by their respective enzymes Were tested using the hairpin substrate shown in FIG. 7 under the conditions described.   DNAPEcl and DNAP Klenow were obtained from Promega Corporation. Pyrococca Pyrococcus furious ["Pfu", Bargseid et al., Strategies 4: 3 4 (1991)] was from Stratagene. Thermococcus litorari (Trm), Vent (exo-), Perler et al., Proc. Natl. Aca d. Sci. USA 89: 5577 (1992)] was from New England Biolabs. Was. Thermus flavus ["Tfl", Kaledin et al., Biokhimiya 4 6: 1576 (1981)] is from Epicentre Technologies and Thermus thermophilus ["Tth", Carballeira et al., Biotec hni-ques 9: 276 (1990); Myers et al., Biochem. 30: 7661 (1991)] is U.S. Bioc from h-emicals.   Buffer supplied by the manufacturer for primer-dependent reactions, or 10 mM Tris Cl, pH 8.5, 1.5 mM MgCl_Two, And 0.5 units of it using 20 mM KCl Each DNA polymerase was assayed in a 20 μl reaction. Enzyme reaction mixture Was maintained at 72 ° C. before the addition of.   FIG. 12 is an autoradiogram recording the results of these tests. FIG. 1 shows the endonuclease reaction of DNAP of some thermophilic bacteria. Prime the reaction solution Incubate at 55 ° C for 10 minutes in the presence of a primer or 72 ° C for 30 minutes in the absence of primer. The product was resolved by denaturing polyacrylamide gel electrophoresis. Product Indicates the length (unit: number of nucleotides). FIG.12B shows DNAPEcl 5 ′ nuclease. 1 shows endonucleolytic cleavage. DNAPEcl reaction solution and The DNAP Klenow reaction was incubated at 37 ° C. for 5 minutes. DNAPEcl lane (it (In the presence and absence of primers, respectively) Note the optical bands of the cleavage products at 11 and 11 nucleotides. Also, FIG. The DNAPTaq reaction in the presence (+) or absence (-) of the immer is shown. These reactions Performed in 50 mM and 20 mM KCl, respectively, and incubated at 55 ° C. for 10 minutes.   Referring to FIG. 12A, the eubacteria Termus thermophilus and Termus fura DNAP from bush is in the same position as DNAPTaq in both the presence and absence of primer To cleave the substrate. In contrast, the archaebacteria Pyrococcus furiosus and Thermoco DNAP from C. litoralis cleaves substrates endonucleolytically Can not. From Pyrococcus frious and Thermococcus litoralis DNAP shares little sequence homology with eubacterial enzymes (Ito et al., Nucl. Acids Res. 19: 4045 (1991); Mathur et al., Nucl. Acids Res. 19: 6952 (1991); see also Perler et al. See). Referring to FIG.12B, DNAPEcl also cleaves the substrate, but the resulting cleavage The product is difficult to detect unless the 3 'endonuclease is suppressed. DNAPEcl and The amino acid sequences of the 5 'nuclease domains of DNAPTaq and DNAPTaq are approximately 38% homologous (Gelf and, supra).   The 5 'nuclease domain of DNAPTaq is the gene 6 of bacteriophage T7. Shares about 19% homology with the 5 'exonuclease encoded by [Dunn et al. J. Mol. Biol. 166: 477 (1983)]. This nucleus is not covalently linked to the DNAP polymerization domain Rease is a site cleaved by the above 5 ′ nuclease in the absence of an added primer DNA can be cleaved endonucleolytically at the same or the same site as You.   C. Trans cleavage   The ability of 5 'nucleases to be efficiently cleaved at specific sequences is described below. Demonstrated in experiments. Partially complementary oligonucleotides called "pilot oligonucleotides" The gonucleotide hybridized with the sequence at the desired position for cleavage. Pyro The non-complementary portion of the tri-oligonucleotide gives a structure similar to the 3 'arm of the template ( On the other hand, the 5 ′ region of the substrate strand became a 5 ′ arm (see FIG. 7). pie The lot folds itself to produce a short hairpin with a stabilized tetra-loop The primer was prepared by designing the 3 'region of the pilot ntao et al., Nucl.Acids Res. 19: 5901 (1991)]. Two types of pilot oligonucleotides The tide is shown in FIG. 13A. Oligonucleotides 19-12 (SEQ ID NO: 18) and 30-12 (sequence No. 19) is 31 or 42 nucleotides in length, respectively. However However, oligonucleotides 19-12 (SEQ ID NO: 18) and 34-19 (SEQ ID NO: 19) It has only 19 and 30 nucleotides, respectively, which are Complementary to different sequences in the land. About 50 ° C for pilot oligonucleotide (19-12) and calculated to melt their complement at about 75 ° C (30-12). Both The other pilots have 12 nucleotides at their 3 'end, and these Acts as a 3 'arm to attach the primer.   To demonstrate that cleavage can be induced by pilot oligonucleotides We have found that single-stranded standards in the presence of two potential pilot oligonucleotides Target DNA was incubated with DNAPTaq. Target and pilot nucleic acids The trans-cleavage reaction, which is not covalently bound, is 0.01 pmole in 20 μl of the same buffer. Single end labeled substrate DNA, 1 unit of DNAPTaq and 5 pmole of pilot Including lignonucleotides. Incubate these components for 1 minute at 95 ° C Denature the PCR-generated double-stranded substrate DNA to match the temperature of the reaction. Decreased to the final incubation temperature. Oligonucleotides 30-12 and 19- 12 is a group that is 85 nucleotides and 27 nucleotides from the 5 'end of the target strand Hybrids can form in regions of quality DNA.   FIG. 23 shows the complete 206-mer sequence (SEQ ID NO: 26). 206-mer was prepared by PCR . M13 / pUC from New England Biolabs (catalog numbers 1233 and 1224, respectively)  The 24-mer reverse sequence (-48) primer and M13 / pUC sequence (-47) primer were PGEM3z (f +) plasmid vector (Promega Corp.) as a template (10 ng) containing (50 pmole each). The PCR conditions were as follows. 100 μl 1X PCR buffer (20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl_Two, 0.05% Tween-20 and 0.05 % NP-40) and 50 μM of each dNTP in 50 mM KCl and 2.5 units of Taq DNA Polymerase. 35 cycles of reaction at 95 ° C for 45 seconds, 63 ° C for 45 seconds, then 72 ° C for 75 seconds did. After cycling, the reaction was terminated by incubation at 72 ° C for 5 minutes. Was. The resulting fragment was purified to 6% in 0.5X TBE (45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA). Purified by electrophoresis on polyacrylamide gel (29: 1 cross-linking), ethidium bromide Visualized by chromium staining or autoradiography, excised from the gel, and passively Eluted by diffusion and concentrated by ethanol precipitation.   Cleavage of the substrate DNA occurs at 50 ° C in the presence of the pilot oligonucleotides 19-12. This (FIG. 13B, lanes 1 and 7) did not occur at 75 ° C. (lanes 4 and 7). And 10). In the presence of oligonucleotide 30-12, cleavage was observed at both temperatures . Even at 50 ° C, accidental structures in the substrate allow primer-independent cleavage in the absence of KCl Even when activated (FIG. 13B, lane 9), the cleavage was in the absence of added oligonucleotide. It did not occur below (lanes 3, 6 and 12) or at about 80 ° C. Complementary to substrate DNA sex Non-specific oligonucleotides with no 50 ° C in the absence or presence of 50 mM KCl Did not induce cleavage (lanes 13 and 14). Thus, the specificity of the cleavage reaction is It can be adjusted by the degree of complementarity to the substrate and the conditions of incubation.   D. Cleavage of RNA   The shortened RNA version of the sequence used in the trans cleavage It was tested for its ability to serve as a substrate in. RNA is Cleavage at expected position in reactions dependent on the presence of gonucleotides . [Α-³²[P] RNA substrates produced by T7 RNA polymerase in the presence of UTP This corresponds to the truncated form of the DNA substrate used in FIG. 13B. The reaction conditions were the above DNA The conditions were the same as for the quality, 50 mM KCl was used. Incubation at 55 ° C For 40 minutes. The pilot oligonucleotide used was 30-0 (SEQ ID NO: 20). ) And shown in FIG. 14A.   FIG. 14B shows the results of the cleavage reaction. The reaction was performed with DNAPTaq or as shown in FIG. Performed in the presence or absence of the pilot oligonucleotide.   Strikingly, in the case of RNA cleavage, the 3 'arm is the pilot oligonucleotide. Not required for This cleavage occurs several times along a 30 base pair long RNA-DNA duplex. It is thought that RNaseH is expected to cleave RNA at certain positions. Absent. The 5 'nuclease of DNAPTaq is located at a single site near the 5' end of the heteroduplex region. It is a structure-specific RNaseH that cleaves RNA.   Surprisingly, oligonucleotides lacking the 3 'arm are effective It can act as a pilot in inducing cleavage. What would be this Such oligonucleotides use natural DNAPs to induce efficient cleavage of DNA targets Because they cannot do it. However, some 5'nuclei of the present invention (For example, clones E, F and G shown in FIG. 16) NA can be cleaved. In other words, the non-extended cleavage structure is a thermostable DNA Not required for specific cleavage by some 5 'nucleases of the invention derived from .   We have found that DNAPTaq in the presence of perfectly complementary primers Cleavage is an RNA template in reactions where DNAPTaq approximates the reverse transcriptase reaction Help explain that DNA oligonucleotides cannot be extended The test was performed to determine whether or not the damage was caused. Another thermophilic DNAP, DNAPTth is Mn⁺⁺Only in the presence of Although RNA can be used as a template, we have Expected not to cleave the RNA in the presence of this cation. Therefore, the present inventors Is Mg⁺⁺Or Mn⁺⁺In the presence of DNAPTaq or DNAPTth in a buffer containing The RNA molecule was incubated with the lot oligonucleotide. Expected As you can see, both enzymes are Mg⁺⁺Cleaved the RNA in the presence of However, DNAPTth Not DNAPTaq is Mn⁺⁺RNA was degraded in the presence of We have found that many DNA When the 5 'nuclease activity of P can contribute to the inability to use RNA as a template Concluded.                                 Example 2            Generation of 5 'nuclease from stable DNA polymerase   Activity, an unwanted side reaction during DNA cleavage in the detection assays of the invention Thermostable nuclease activity while maintaining the thermostable nuclease activity. NA polymerase was generated. The result is a heat that cleaves nucleic acid DNA very specifically. It is a stable polymerase. DNA polymerase A from eubacteria of the genus Termus Type is a wide range of protein sequence identity (DNA analysis software from DNAStar, WI) Using the Lipman-Pearson method at 90% in the polymerization domain) It behaves similarly in both nuclease assays. Therefore, the present inventors Thermus Aquatics (DNAPTaq) and Therms Flabs (DN APTfl) DNA polymerase gene was used. Other eubacterial organisms, even if For example, Termus thermophilus, Termus sp. , Termotoga Maritima, Terumo Sypho Africanus and Bacillus stearothermophilus, a polymer from The lase gene is equally suitable. DNA polymerases from these thermophilic organisms Zes can survive and behave at elevated temperatures, thus increasing the temperature of nucleic acid strands. Can be used in reactions used as a selection tool for heterogeneous hybridization You. The restriction sites used for the following deletion mutagenesis were chosen for convenience. Similar facilities Are available in the Termus thermophilus gene and are Can be used to create structures similar to those of the other type A polymerase genes.   A. Generation of 5 'nuclease structure     1. Modified DNAPTaq gene   The first step is to perform Taq DNA polymerase on the plasmid under the control of an inducible promoter. Was to put the modified gene of ze. The modified Taq polymerase gene is Seared isolated. The Taq DNA polymerase gene was set forth in SEQ ID NOs: 13-14. Using Oligonucleotides as primers, Termus Aquatics, strains Polymerase chain reaction from genomic DNA from YT-l (Lawyer et al., Supra). Amplified. The resulting DNA fragment contains a restriction endonuclease at the 5 'end of the coding sequence. It has a recognition sequence for EcoRI and a BglII sequence at the 3 'end. Cleavage by BglII is B Retain 5 'overhangs or "sticky ends" compatible with ends created by amHI You. The PCR amplified DNA was digested with EcoRI and BamHI. Polymerase gene The 2512 bp fragment containing the coding region was gel purified, and then the Connected to Rasmid.   In one embodiment of the invention, the pT comprising the hybrid trp-lac (tac) promoter Using the TQ18 vector [M.J.R.Stark, Gene 5: 255 (1987)] and shown in FIG. tac The lomotor is under the control of the E. coli lac repressor. Inhibition is the desired growth of bacteria Allow the synthesis of the gene product to be suppressed, at which point Is a specific inducer, isopropyl-bD-thiogalactopyranoside (I PTG) is removed. Such systems slow or inhibit the growth of transformants. Allow expression of foreign proteins that can be stopped.   Bacterial promoters, such as tac, are present on many copy plasmids Sometimes it may not be properly suppressed. Highly toxic proteins such as When placed under the control of a promoter, small amounts of expression that escape control can be harmful to bacteria. possible. In another embodiment of the invention, the synthesis of the cloned gene product is suppressed. Used another alternative for. Bacteria found in the plasmid vector series pET-3 Mutant Taq cloned using a non-bacterial promoter from Teriophage T7 Expressed the remelase gene [Figure 15; Studier and Moffatt, J. Mol. Biol. 189:  113 (1986)]. This promoter initiates transcription by T7 RNA polymerase only. Start. In a suitable strain such as BL21 (DE3) pLYS, the gene for this RNA polymerase Is carried in the bacterial genome under the control of the lac operator. This arrangement is Expression of multiple copy genes (with respect to plasmid) expression of T7 RNA polymerase And has the advantage that this is easily suppressed. What would happen Because it exists in a single copy.   For binding to the pTTQ18 vector (Figure 15), the Taq polymerase PCR product DNA (mutTaq, clone 4B, SEQ ID NO: 21) digested with EcoRI and BglII This fragment was then used under normal “sticky end” conditions [Sambrook et al., Molecular Cloning, Co. ld Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1.63-1.69 (1989) )] To the plasmid vector pTTQ18 at the EcoRI site and the BamHI site. this Expression of the construct is based on the first two residues (Met-Arg) of the native protein Resulting in a translation fusion product that is replaced by three residues (Met-Asn-Ser) The rest of the natural protein will not change. E.coli JM109 Transforms strains and does not allow the growth of bacteria expressing the native protein Placed under incomplete suppression conditions. These plating conditions are set during the amplification process. Including existing mutations such as those arising from the infidelity of Taq polymerase Enables isolation of genes.   Using this amplification / selection protocol, we used the mutated Taq Isolate a clone containing the Rase gene (mutTaq, clone 4B) (shown in Figure 16B) did. The mutant is first detected by its phenotype, in this case crude cell extraction Temperature stable 5 'nuclease activity in the product was normal, but polymerization activity was almost absent. (Less than about 1% of wild-type Taq polymerase activity).   DNA sequence analysis of the recombinant gene reveals that the It has been shown to have changes in the lase domain. From A at nucleotide position 1394 The change to G is at amino acid position 465 (natural nucleic acid sequence and amino acid sequence, SEQ ID NO: 1 and Glu to Gly changes (numbered according to and 4) and The change from A at position 2260 to G causes a change from Gln at position 754 to Arg. I will. The Gln to Gly mutation is in a non-conserved position, and also a Glu to Arg collision. Mutations alter amino acids conserved in virtually all known type A polymerases This latter mutation probably reduces the synthetic activity of this protein. Is a mutation that causes The nucleotide sequence of the clone 4B construct (FIG. This is shown in SEQ ID NO: 21. The corresponding encoded by the nucleotide sequence of SEQ ID NO: 21 The amino acid sequence is shown in SEQ ID NO: 72.   Next, derivatives of the DNAPTaq construct were made from the mutTaq gene, thus This, apart from those other changes, unless these particular regions are deleted Have all of these amino acid substitutions. These mutation sites are shown in FIG. These positions are indicated by black boxes. In FIG. 16, use the display "3'Exo" Of 3 'exonuclease activity associated with type A polymerase not present in NAPTaq Indicates the position. All constructs except the genes shown in FIGS. 16E, F and G were Made in a reactor.   The cloning vectors used for the genes in FIGS. From the pET-3 series. This vector series is downstream of the T7 promoter. Although it has only a BamHI site for cloning, the series has three leading Includes variants that allow cloning into any of the frames. The PCR product For roning, a variant designated pET-3c was used (FIG. 17). The vector Was digested with BamHI, dephosphorylated with calf intestinal phosphatase, and digested with DNAPEcl and dNTPs. Cohesive ends were filled in using Klenow fragment. Mutant Taq shown in FIG.16B The DNAP (mutTaq, clone 4B) gene was digested with EcoRI and SalI into pTTQ18 And filled in the "sticky ends" as done with the vector. That fragment Under standard blunt-end conditions (Sambrook et al., Molecular Cloning, supra). And transforming the construct into the BL21 (DE3) pLYS strain of E. coli, Screen isolates and ligate genes in proper orientation to promoter The isolated isolate was identified. This construct produces another translation fusion product, in which case The first two amino acids (Met-Arg) of DNAPTaq are 13 from the vector + PCR primer (Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg -Ile-Asn-Ser) (SEQ ID NO: 23).   Our goal is to lack the ability to synthesize DNA but have a 5 'nuclease activity. It was to create an enzyme that possesses the ability to retain the ability to cleave nucleic acids. DNA The effect of template synthesis using primers is actually a series of regulated phenomena. In this way, disturb one phenomenon without affecting other phenomena to disable DNA synthesis Can be These steps include primer recognition and binding, dNTPs Catalysis of phosphodiester bonds between bonds and nucleotides, It is not limited to these. Some of the amino acids in the polymerization domain of DNAPEcI But the exact mechanism has not yet been fully elucidated. No.   One way to disrupt the polymerization ability of DNA polymerases is with their proteins Deleting all or part of the gene segment encoding the domain, Or otherwise, the gene will not be able to create a fully polymerized domain It is to do. Each mutant enzyme is stable and extracellular and extracellular. And solubility may differ from each other. For example, the 5 'nuclease of DNAPEcI Main (which is released in an active form from the polymerization domain by mild proteolysis [Setlow and Kornberg, J. Biol. Chem. 247: 232 (1972)]). Thermus nuclease domain, when treated similarly, has reduced solubility. , Its cleavage activity is often lost.   Using the mutant gene shown in FIG. 16B as starting material, several deletion constructs I made things. All cloning techniques are standard (Sambrook et al., Supra). The following is a brief summary.   Figure 16C: The mutTaq construct was digested with PstI and the polymerase coding region as indicated. Cut once in the region and at the multiple cloning site of the vector just downstream of the gene. Disconnect. After release of the fragment between these two sites, the vector was religated and , Causes an 894-nucleotide deletion and a 40 stop codon downstream of the linkage in frame Leotide was added. The nucleotide sequence of this 5 'nuclease (clone 4C) is Shown in column number 9. The corresponding DNA encoded by the nucleotide sequence of SEQ ID NO: 9 The amino acid sequence is shown in SEQ ID NO: 73.   Figure 16D: Digestion of the mutTaq construct with NheI, which cuts once at position 2047 in the gene I do. Fill in the resulting 4 nucleotide 5 ′ overhang end as described above, The blunt ends were ligated again. The resulting 4-nucleotide insertion is the reading frame To terminate translation 10 amino acids downstream of the mutation. This 5 'nucleus SEQ ID NO: 10 shows the nucleotide sequence of Ze (clone 4D). SEQ ID NO: 10 The corresponding amino acid sequence encoded by the otide sequence is shown in SEQ ID NO: 74.   Figure 16E: Cleavage of the complete mutTaq gene from pTTQ18 using EcoRI and SalI And cloned into pET-3c as described above. This clone is shown in FIG. Digested with BstXI and XcmI at unique sites located as shown. DNAP Treated with Klenow fragment of Ec1 and dNTP, trimmed 3 'overhangs of both sites and flattened The ends were smooth. These blunt ends were ligated together and an 1540 nucleotide out- This resulted in an out-of-frame deletion. In-frame stop codon Occurs after 18 triplets of the binding site. This 5 'nuclease (clone 4E) The nucleotide sequence is shown in SEQ ID NO: 11. The corresponding amino acid sequence is shown in SEQ ID NO: 75]. Arrange an appropriate leader sequence. Shown in SEQ ID NO: 24 [corresponding DNA encoded by the nucleotide sequence of SEQ ID NO: 24 The amino acid sequence is shown in SEQ ID NO: 76]. It is also Cleavase^TMCalled BX enzyme.   Figure 16F: Cleavage of the complete mutTaq gene from pTTQ18 using EcoRI and SalI And cloned into pET-3c as described above. Copy this clone as shown Digested with BstXI and BamHI at a unique site located at The DNA is transferred to DNAPEc1 And Klenow fragment of dNTP, trimmed 3 'overhangs at both sites and blunt-ended On the other hand, the 5 'overhang of the BamHI site was filled in to obtain blunt ends. these The ends were ligated together resulting in an in-frame deletion of 903 nucleotides. This SEQ ID NO: 12 shows the nucleotide sequence of 5 'nuclease (clone 4F). It Also Cleavase^TMCalled BB enzyme. The nucleotide sequence of SEQ ID NO: 12 The corresponding amino acid sequence is shown in SEQ ID NO: 77.   FIG. 16G: This polymerase is a variant of the polymerase shown in FIG. 16E. It was cloned into the plasmid vector pET-21 (Novagen). In this vector Non-bacterial promoter derived from bacteriophage T7 is T7 RNA polymerase Transcription will not start unless it depends. Studier and Moffatt, see above. . In a suitable strain such as (DES) pLYS, the gene for this RNA polymerase is lac-operated. Carried by the bacterial genome under the control of This arrangement is a multiple copy gene Expression (on the plasmid) is completely dependent on T7 RNA polymerase expression, This has the advantage that this is easily suppressed. Because it exists in a single copy Because there is. The expression of these mutant genes is The toxicity of the expressed protein to the host cell as it occurs under the The potential problem is not of concern.   Also, the pET-21 vector is added to the carboxy terminus of the expressed protein. It is characterized by a "His-tag", which is a stretch of six consecutive histidine residues. Then obtained Protein is immobilized on Ni⁺⁺Uses commercial (Novagen) column resin containing ions And purified in a single step by metal chelation chromatography. 2.5ml mosquito The rum is reusable, natural conditions or denatured (guanidine-HCl or urea) Under conditions, up to 20 mg of the target protein can be bound.   Transform E. coli (DES) pLYS cells with the above constructs using standard transformation techniques. And used to inoculate a normal growth medium (eg, Luria-Bertani broth). You. Induction of T7 RNA polymerase production during log phase growth by addition of IPTG, Incubate for 12-17 hours. Remove aliquots of the culture before and after induction, The protein is tested by SDS-PAGE. Foreign protein is about 3 of cellular proteins If they occupy ~ 5% and do not co-migrate with any of the major host protein bands, Coomassie blue staining allows visualization of foreign proteins. Main host Proteins that co-migrate with the protein must be fully loaded to be seen at this stage of the analysis. It must be expressed as 10% or more of the protein.   Some mutant proteins are taken up by the cells into inclusion bodies. These are fine Formed in the cytoplasm when bacteria were created to express high levels of foreign proteins Granules which are purified from the crude lysate and analyzed by SDS-PAGE. Their protein content can be measured. Cloned protein is present in inclusion bodies Must be released to assess cleavage and polymerase activity. It is necessary. Different solubilization methods are suitable for different proteins, and different methods are known. Have been. See, e.g., Builder & Ogez, U.S. Patent No. 4,511,502 (1985); U.S. Patent No. 4,518,526 (1985); Olson & Pai U.S. Patent No. 4,511,503 (1985); Jon See US Patent No. 4,512,922 (1985) to es et al. All of these are for reference Included herein.   The solubilized protein is then processed as described above according to the manufacturer's instructions (Novagen). Ni⁺⁺Purify on column. Washed protein with imidazole competitor (1M) Elute from the column with a combination of salts (0.5M NaCl), dialyze and change the buffer. Regenerate denatured protein. A typical harvest is about 20 μg / ml starting culture Of the specific protein. DNAP mutant was converted to Cleavase^TMBN) Yeast And the sequence is shown in SEQ ID NO: 25. By the nucleotide sequence of SEQ ID NO: 25 The encoded corresponding amino acid sequence is shown in SEQ ID NO: 78.   2. Modified DNAPTfl gene   The T. flava DNA polymerase gene obtained from the ATCC Strain AT-62 (ATCC 33923). This strain was isolated from Akhmetzjanov and Vakhitov. Differs from the T. flavus used to generate the sequences published by Have a restriction map. The published sequence is shown in SEQ ID NO: 2. T. Flavus AT-62 Sequence data for the DNA polymerase gene from the strain were not published .   Used to amplify the T. aquatic DNA polymerase gene (SEQ ID NOs: 13-14) Genomic DNA from T. flavus was amplified using the same primers. About 2 The 500 base pair PCR fragment was digested with EcoRI and BamHI. DNAPEc1 Klenow with protruding end Fragments and blunted with dNTPs. About 1800 bases obtained, including the N-terminal coding region The pair fragment was ligated to pET-3c as described above. This construct, clone 5B, is Shown in The wild-type T. flavus DNA polymerase gene is shown in FIG. 18A. Fig. 18 Medium, using the label “3'Exo” to associate with type A polymerase not present in DNAPTfl 3 indicates the position of the 3 ′ exonuclease activity. 5B clone cloned into pET-3c DNAPTaq clones 4E and F have the same leader amino acids. Translation stopped It is not known exactly where it occurs, but the vector is It has a strong transcription termination signal immediately downstream of the position.   B. Propagation and induction of transformed cells   Bacterial cells are transformed with the above constructs using conventional transformation techniques and It was used to inoculate 2 ml of growth medium (eg Luria-Bertani broth). Get Cultures are incubated as appropriate for the particular strain used, and Induced if needed. For all of the constructs shown in FIGS. 16 and 18, For this, the culture was grown to an optical density of 0.5 OD (wavelength 600 nm).   Cultures were brought to a final concentration of 0.4 mM IPTG to induce expression of the cloned gene. The incubation was continued for 12-17 hours. 50 μl aliquots of each culture The salt was removed before and after induction and sodium dodecyl sulfate-polyacrylamide gel was added. 20 μl of normal gel loading buffer for gel electrophoresis (SDS-PAGE) . Foreign proteins make up about 3-5% of cellular proteins and are the major E. coli proteins If not co-migrated with any of the bands, subsequent staining with Coomassie blue (S ambrook et al., supra) allow visualization of foreign proteins. Major host tampa Proteins that co-migrate with the protein must have total protein to be seen at this stage of the analysis. Must be expressed as at least 10% of the quality.   C. Thermolysis and fractionation   Heat the crude bacterial cell extract to denature less stable E. coli proteins. And by precipitation, the expressed thermostable protein, i.e., the 5 ' The ase was isolated. Next, precipitate the precipitated E. coli protein together with other cell debris. And removed by centrifugation. 1.7 ml of culture solution at 12,000-14,000 rpm for 30-60 seconds Pelletized by centrifugation. After removing the supernatant, the cells were washed with buffer A (50 mM Tris- HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA). Centrifuge and then resuspend in 80 μl buffer A with 4 mg / ml lysozyme I let it. The cells are incubated at room temperature for 15 minutes, then buffer B (10 mM Tris-HCl PH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM PMSF, 0.5% Tween-20, 0.5% Nonidet-P40 ) 80 μl.   This mixture was incubated at 75 ° C for 1 hour to denature the host protein and precipitate. I let it. The cell extract was centrifuged at 14,000 rpm for 15 minutes at 4 ° C. Transferred to tube. Aliquots of 0.5-1 μl of this supernatant were used for each test reaction. Use directly and extract 7 μl by electrophoretic analysis as described above. The protein content of the product was measured. Original recombinant Taq DNA polymerase [Englke, A nal. Biochem 191: 396 (1990)], and the two-point mutant protein shown in FIG. Both are soluble and active at this point.   The foreign protein is subjected to heat treatment for the uptake of the foreign protein by the cells into the inclusion bodies. It may not be detected after processing. These allow bacteria to produce high levels of foreign proteins. Granules that form in the cytoplasm when created to express The lysates can be purified and analyzed by SDS PAGE to determine their protein content. Many methods are described in the literature, one approach is described below.   D. Isolation and solubilization of inclusion bodies   Small cultures were grown and induced as described above. 1.7 ml aliquots in a short time Pellet by centrifugation of bacterial cells in lysis buffer (50 mM Tris-HCl, pH 8.0 , 0.1 mM EDTA, 100 mM NaCl). 20 μM PMSF 2.5 μl 0 Lysozyme was added to a final concentration of 0.5 mM and lysozyme was added to a concentration of 1.0 mg / ml. Chamber cells Incubate for 20 minutes at room temperature and add 1 mg / ml of deoxycholic acid (1 μl of 100 mg / ml solution). ) And incubate the mixture at 37 ° C for about 15 minutes or until viscous. I did it. DNAse I is added to 10 μg / ml and the mixture is allowed to stand at room temperature for about 30 minutes or Incubate until it is no longer viscous.   From this mixture, inclusion bodies were recovered by centrifugation at 14,000 rpm for 15 minutes at 4 ° C. The supernatant was discarded. The pellet was dissolved in 10 mM EDTA (pH 8.0) and 0.5% Triton X-100. Resuspended in 100 μl of digestion buffer. After 5 minutes at room temperature, remove the inclusion bodies as described above. Pelleted and saved the supernatant for subsequent analysis. Fill the inclusion body with 50 μl of distilled water And re-suspend in 5 μl of SDS gel loading buffer (which dissolves inclusion bodies). And an aliquot of the supernatant was analyzed by electrophoresis.   If the cloned protein is present in inclusion bodies, cleavage activity and polymer Release it to evaluate the activity of the enzyme. Solubilization method is compatible with specific activity Need to be gender. Different solubilization methods are suitable for different proteins, Is described in Molecular Cloning (Sambrook et al., Supra). Less than Is an improved method that we have used in some of our isolates.   20 μl of the inclusion body-water suspension was centrifuged at 14,000 rpm for 4 minutes at room temperature. And pelleted the supernatant. Pellet 2M urine to further clean the inclusions Resuspended in 20 μl of lysis buffer containing phosphoric acid and incubated for 1 hour at room temperature . The washed inclusion bodies were then resuspended in 2 μl of lysis buffer containing 8M urea. Seal As the deposit dissolved, the solution became visually clear. Undissolved crushed material at room temperature By centrifugation at 14,000 rpm for 4 minutes at room temperature. Moved to a tube.   To reduce urea concentration, extract_TwoPO_FourIn water. 180 μl of 50 mM K H_TwoPO_Four(pH 9.5) A new tube containing 1 mM EDTA and 50 mM NaCl was prepared. Lottery A 2 μl aliquot of the output was added and vortexed quickly to mix. Of the extract This process was repeated until all had been added in a total of 10 additions. That blend The mixture is left at room temperature for 15 minutes, during which time some precipitation often occurs. Precipitate at room temperature Remove by centrifugation at 14,000 rpm for 15 minutes in a Moved to KH_TwoPO_Four200 μl of protein in solution is saturated (NH_Four)_TwoSO_FourAdd 140-200μl And the resulting mixture is about 41% -50% saturated (NH_Four)_TwoSO_FourMet. That The mixture was cooled on ice for 30 minutes to precipitate the protein, and then the protein was allowed to stand at room temperature for 4 hours. Collected by centrifugation at 14,000 rpm for minutes. Discard supernatant and pellet Buffer C (20 mM HEPES (pH 7.9), 1 mM EDTA, 0.5% PMSF, 25 mM KCl and each Was dissolved in 20 μl of 0.5% Tween-20 and Nonidet P40). Again with the protein solution Pellet the insoluble material by centrifugation for 4 minutes and remove the supernatant to a fresh tube. Separated. This method is prepared by analyzing 1-4 μl by SDS-PAGE. The protein content of the prepared extract was visualized. 0.5 to 1 μl of extract Tested in cleavage and polymerization assays as described.   E. FIG. Protein analysis for nuclease presence and synthetic activity   The 5 ′ nuclease described above and shown in FIGS. 16 and 18 was analyzed by the following method. Was.   1. Structure-specific nuclease assay   Testing candidate modified polymerases for their ability to catalyze structure-specific cleavage And tested for 5 'nuclease activity. As used herein The term “cleavage structure” refers to a group for cleavage of DNAP by 5 ′ nuclease activity. Quality nucleic acid construct.   The polymerase is exposed to a test complex having the structure shown in FIG. 5 'Nuclear Testing for zease activity involves three reactions. 1) Perform primer-specific cleavage (Figure 19B) U. What makes it relatively insensitive to changes in salt concentration in the reaction Therefore, it can be performed under any solute conditions required for the activity of the modifying enzyme. This is generally the same as the conditions suitable for an unmodified polymerase. 2) Similar ply A buffer that allows primer-independent cleavage of the mer-specific cleavage, i.e., the enzyme Performed in low salt buffer to demonstrate that 3) Perform primer-independent cleavage (FIG. 19A) in the same low salt buffer.   A branched duplex is formed between the substrate strand and the template strand shown in FIG. Used herein The term “substrate strand” refers to the cleavage at which cleavage mediated by 5 ′ nuclease activity occurs. Nucleic acid chain. Substrate chain can be used as a substrate for 5 'nuclease cleavage Always shown as the upper strand in the complex (Figure 19). As used herein, "template strand" The term "is at least partially complementary to the substrate strand and anneals to the substrate strand. Means the strand of the nucleic acid that forms the cleavage structure. The template strand is the lower strand of the branch cleavage structure. (Figure 19). As when primer-dependent cleavage is tested, Add primer (short oligonucleotide 19-30 nucleotides in length) to complex If so, it is designed to anneal to the 3 'arm of the template strand (Figure 19B). To the reaction If the polymerase used has synthetic activity, such primers It should extend along the chain.   Cleavage structures may be made as single-stranded hairpin molecules, which target the 3 'end and And the pilot 5 'end joined as a loop as shown in FIG. 19E. Ma In addition, these tests require primer oligonucleotides complementary to the 3 'arm. As a result, one can test the sensitivity of the enzyme to the presence of the primer.   The nucleic acids used to form the test cleavage structure can be chemically synthesized or It can be made by conventional recombinant DNA techniques. By the latter method, the hairpin of the molecule Portions clonally duplicate double copies of short DNA segments adjacent to each other but in the opposite direction. Can be made by inserting the vector into a vector. Next, this reverse Short (about 20 nucleotides) unpaired 5 'and 3' arms, including peat Double-stranded fragment containing sufficient flanking sequences to obtain Enzymes lacking 5 'exonuclease (eg, AmpItaq^TMDNA polymerase Stoffel fragment, Vent^TMReleased from vector by PCR performed with DNA polymerase) I can do it.   Test DNA may be labeled with a radioisotope or non-isotopic tag at either end, Can label the interior. Hairpin DNA is synthetic single-stranded or cloned double-stranded Regardless, the DNA is heated before use to melt all duplexes. Cool on ice When formed, the structure shown in FIG. 19E is formed, Stable for a time sufficient to perform.   To test for primer-specific cleavage (reaction 1), Test molecules (typically 1-100 fmole³²P-labeled hairpin molecule) and 10 to 100-fold molar Place excess primer in a buffer known to be compatible with the test enzyme. You. For reaction 2, primer-specific cleavage allows primer-independent cleavage When performed under similar conditions, the same amount of molecule is combined with pH, enzyme stabilizers (eg, bovine blood). Qing aluminin, nonionic surfactant, gelatin) and reducing agent (for example, dithio Buffer used in reaction 1 for threitol, 2-mercaptoethanol) As above, but in a solution in which the monovalent cation salt is replaced by 20 mM KCl. 20mM KCl Has been demonstrated to be optimal for primer-independent cleavage. Works normally in the absence of salt Buffers for enzymes such as DNAPEc1 are not added to achieve this concentration. No. To test for primer-independent cleavage (reaction 3), But not the same amount of test molecule under the same buffer conditions used for reaction 2. Hang on.   All three reactions were then performed with enough enzyme to test complex molar ratio of about 1: 1. Expose to enzymes. Below the temperature allowed by enzyme stability or complex stability Temperature, which is below 80 ° C for enzymes from thermophilic bacteria, allows cleavage Incubate the reaction for a sufficient time (10-60 minutes). Reactions 1, 2 and And 3 products were separated by denaturing polyacrylamide gel electrophoresis. Visualize by ography or an equivalent method appropriate to the labeling system used. another Labeling systems include chemiluminescence detection, silver or other stains, And probing. The presence of the cleavage product indicates that the uncleaved test structure Indicated by the presence of molecules that migrate at lower molecular weights. These cleavage products are 4 shows that the polymerase has structure-specific 5 'nuclease activity.   Modified DNA polymerase substantially matches the 5 'nuclease activity of native DNA polymerase To determine whether they have the same 5 'nuclease activity, Naturally, the results obtained from these tests performed with DNA polymerase are compared. "Substantially the same 5 'nuclease activity" refers to modified polymerases and native polymer Both are the sameStyleMeans to cleave the test molecule. Modified polymerer Is the same as natural DNA polymerasespeedIt is not necessary to cleave at   Some enzymes or enzyme preparations are functional under the above cleavage conditions and are 5 ' Activities of other related activities or contaminants that may interfere with nuclease detection (con taminating activity). Reaction conditions include substrate degradation or 5'nuclease To avoid cleavage of the enzyme and other masking of its products, these other It can be improved in consideration of the activity. For example, E. coli DNA polymerase I (Pol I) In addition to the polymerase and 5 'nuclease activities of DNA, the DNA is separated in the 3' to 5 'direction. It has a recognizable 3 'exonuclease activity. Therefore, the molecule in FIG. 3 'exonuclease activity forms a pair when exposed to this polymerase The 3 'arm, which has not been removed, is promptly removed and required as a substrate for 5' exonuclease cleavage. The resulting branched structure is destroyed and no cleavage is detected. PolI that cleaves its structure The true ability is that 3 'exonucleases can change conditions (eg, pH), Phenomena, or when inhibited by the addition of a competitor for its activity. Can be. For the cleavage reaction by Pol I, 500 pmole Addition of a single-stranded competitor oligonucleotide results in the release of 5 'exonuclease on the 5' arm. Without interfering with separation, digestion of the 3 'arm of the FIG. 19E construct is effectively suppressed. Competitor concentration Degree is not critical, but occupies 3 'exonuclease over the duration of the reaction Should be high enough to   Similar degradation of the test molecule is caused by contaminants in the candidate polymerase preparation. May be rubbed. A structure-specific nuclease reaction is performed for candidate nucleases. The purity is determined and overexposure and overexposure of the test substance to the polymerase preparation to be studied are performed. Can be run in several sets to find windows during low exposure You.   The above modified polymerase was tested for 5 'nuclease activity as follows. Tested. Reaction 1 was performed at 10 mM Tris-HCl (pH 8.5), 20 ° C, 1.5 mM MgCl_TwoAnd 50 mM KCl In reaction 2, the KCl concentration was reduced to 20 mM. Reactions 1 and 2 Then, 10 fmole of the test substrate molecule shown in FIG. And an extract containing the modified polymerase (prepared as described above) Combined with l. This mixture was then incubated at 55 ° C. for 10 minutes. Tested For all of the mutant polymerases, these conditions are sufficient to obtain complete cleavage. Met. When the molecule shown in FIG. The isolated 5 'fragment was purified with 20% polyacrylamide gel containing 7M urea in 0.5X TBE buffer. (19: 1 cross-linking). Clones 4C-F and 5B contain unmodified DNA polymerase It showed a structure-specific cleavage comparable to that of lyse. In addition, clones 4E, 4F and 4G Has the added ability to cleave DNA in the absence of a 3 'arm as described above. A representative cleavage reaction is shown in FIG.   For the reaction shown in FIG. 20, mutant polymerase clone 4E (Taq mutant) and And 5B (Tfl mutants) have their ability to cleave the hairpin substrate molecule shown in FIG. Tested for force. Substrate molecule at 5 'end³²P-labeled. 10fmole heat denatured, End-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1) or 0.5 μl of 4e Or 5b extract (FIG. 20, lanes 2-7, extract prepared as above) Was added to 10 mM Tris-HCl (pH 8.5), 50 mM KCl and 1.5 mM MgCl_TwoIn a buffer containing And mixed together. The final reaction volume was 10 μl. Shown in lanes 4 and 7 The reaction also contains 50 μM of each dNTP. Lanes 3, 4, 6, and 7 The reaction mixture shown in (1) was prepared using 0.2 μM of the primer oligonucleotide (with the 3 ′ arm of the substrate). (Complementary and shown in FIG. 19E). The reaction was incubated at 55 ° C for 4 minutes . The reaction was stopped by the addition of 8 μl stop solution per 10 μl reaction volume. Next, The samples were applied to a 12% denaturing acrylamide gel. After electrophoresis, run the gel Autoradiography. FIG. 20 shows that clones 4E and 5B It shows that it shows cleavage activity similar to the activity. Certain cleavages occur in the absence of primer Note that this occurs during these reactions. Structure used here (Figure 19E) Long hairpin structures, such as those used for cleavage reactions performed in a buffer containing 50 mM KCl. When used, low levels of primer-independent cleavage are seen. Higher concentration of KCl Inhibits, but does not eliminate, this primer-independent cleavage under these conditions.   2. Assay for synthetic activity   Modification enzyme or proteolysis can be achieved by adding the modification enzyme to the assay system. Evaluate the performance of the fragments. In this assay system, the primer anneals to the template, DNA synthesis is catalyzed by the added enzyme. Many standard laboratory techniques use such techniques. Essays are used. For example, nick translation and enzyme sequencing Involves extension of the primer along the DNA template by the polymerase molecule.   A preferred assay for measuring the synthetic activity of the modifying enzyme is an oligonucleotide. Primer to a single-stranded DNA template (eg, bacteriophage M13 DNA). To remove the primer / template duplex from the modified polymerase, deoxynucleotide. Suitable for sidotriphosphate (dNTP) and unmodified or natural enzymes Incubate in the presence of known buffers and salts. Primer extension (By denaturing gel electrophoresis) or dNTP incorporation (acid precipitation or chromatography) Is an indicator of active polymerase. Isotopically labeled or non-isotopic Isotopic labels are included as primers or dNTPs to facilitate detection of polymerization products Is preferred. Synthetic activity depends on free nucleotides incorporated into growing DNA strands. Quantified as an amount and taken up per unit time under specific reaction conditions Is represented by   Representative results of the assay for synthetic activity are shown in FIG. Mutant DNAPTaq black The synthetic activity of the protein 4B-F was tested as follows. Master mixing of the following buffers I made things. 12X PCR buffer, 50 μM each of dGTP, dATP and dTTP, 5 μM dCTP and 0.125 μM 600 Ci / mmol α-³²P-dCTP. This mixture to its final volume Before adjustment, it was divided into two equal aliquots. One side of this aliquot Was added to 50 μl of water to reach the above concentration. On the other hand, single-stranded M13mp18 DNA (About 2.5 pmole or 0.05 μM final concentration) and 250 pmole of M13 sequencing primer ( (5 μM final concentration) and distilled water to a final volume of 50 μl. Each mosquito The kutelle was warmed to 75 ° C. for 5 minutes and then cooled to room temperature. As a result, a mixture containing DNA In the mix, the primers were annealed to the DNA.   For each assay, add 4 μl of the cocktail containing DNA as described 1 μl of the mutant polymerase prepared as above or 1 unit of DNAPTaq (Perki dH including n Elmer)_TwoO1 μl. “No DNA” control in the presence of DNAPTaq (Fig. 21, lane 1) and a pair of "no enzyme" using water instead of enzyme. Illumination (lane 2). Mix each reaction, then add 5 minutes at room temperature (about 22 ° C). Incubate at 55 ° C for 2 minutes, then at 72 ° C for 2 minutes. Was. Perform this stage incubation to ensure that all that may have an optimal temperature Polymerization in the mutant of was detected. After the final incubation, Was quickly centrifuged to collect the condensate and placed on ice. 1 μl of each reaction solution Polyethyleneimine (PEI) cellulose thin layer chromatography The sample was spotted as an origin at a position 1.5 cm away from the plate and dried. Buffer front Chromatography plate is 0.75M NaH until approximately 9cm above origin_TwoPO_Four( pH 3.5). Dry the plate, wrap it in plastic wrap, Marked with g and exposed to X-ray film. Stay at the first spot Detected incorporation as a count, while unincorporated nucleotides Was transferred from the origin by this salt solution.   Comparison of the location of the counts in the two control lanes indicates the polymerization activity in the mutant preparation. The lack of gender was confirmed. Of the modified DNAPTaq clones, only clone 4B is shown in FIG. To retain the remaining synthetic activity.                                 Example 3          5 'nuclease derived from thermostable DNA polymerase          Can specifically cleave short hairpin structures   Cleavage of the hairpin structure, resulting in a cleaved hairpin structure suitable as a detection molecule The ability of the 5 'nuclease to make bodies was tested. Hairpin test molecule structure and The sequence is shown in FIG. 22A (SEQ ID NO: 15). On the 3 'arm of the hairpin test molecule, its complementary arrangement The oligonucleotides annealed to the column (primer in FIG. 22A, SEQ ID NO: 22) Show. The hairpin test molecule is a labeled T7 promoter in the polymerase chain reaction. Using primers,³²Single end labeled with P. Label is 5 for hairpin test molecule 'Residing on the arm, represented by an asterisk in FIG. 22A.   10 fmole of the heat-denatured end-labeled hairpin test molecule was added to 0.2 μM primer Oligonucleotide (complementary to the 3 'arm of the hairpin), 50 μM of each dNTP and 0.5 unit Extract containing DNAPTaq (Perkin Elmer) or 5 'nuclease (as described above) 0.5 μl of 10 mM Tris-HCl (pH 8.5), 50 mM KCl and 1.5 mM MgCl_Two Was added to a total volume of 10 μl of a buffer solution containing the to perform a cleavage reaction. Lane 3 The reactions shown in 5, 5 and 7 were performed in the absence of dNTP.   The reaction was incubated at 55 ° C. for 4 minutes. 8 μl reaction per 10 μl reaction volume Stopped at 55 ° C. by adding stop solution. Do not heat the sample; Liacrylamide gel (10% polyacrylamide, 19: 1 cross-linking, and 7M urea IX TBE [89 mM Tris-borate (pH 8.3), 2.8 mM EDTA]). Sump A single-stranded hairpin molecule and a re-doubled uncleaved hairpin without heating The molecules were separated.   FIG. 22B shows the oligonucleotide being annealed to the single-stranded 3 ′ arm of the hairpin and cleaving An altered polymer that lacks detectable synthetic activity when yielding a single species of product It shows that the enzyme cleaves the hairpin structure (FIG. 22B, lanes 3 and 4). The 5 'nuclease, such as clone 4D, shown in lanes 3 and 4, was in the presence of dNTPs. Even produce a single cleavage product. Residual synthetic activity (less than 1% of wild-type activity) The 5 'nuclease that retains a variety of cleavage products. What is the polymer Lase extends the oligonucleotide annealed to the 3 'arm of the hairpin, which Can move the cleavage site (clone 4B, lane 5 And 6). Natural DNA Taq is more efficient than a mutant polymerase that retains residual synthetic activity. High levels in native polymerase in the presence of dNTPs The hairpin structure is further converted to the double-stranded form by the synthetic activity of.                                 Example 4                           Cleavage of linear nucleic acid substrates   Thus, natural (ie, “wild-type”) thermostable DNA polymerases are Hairpin structures can be cleaved in a fashion, and this finding is a success in detection assays. It should be clear that it can be applied to In this example, the mutant DNAP of the present invention Are tested against three different cleavage structures shown in FIG. 24A. Structure in FIG.24A 1 is a simple single-stranded 206-mer (the preparation and sequence information of which is described above) ). Structures 2 and 3 are double-stranded. Structure 2 is shown in FIG. 13A (bottom). Structure 3 is the same hairpin structure, while structure 3 has the hairpin portion of structure 2 removed. Have been.   The cleavage reaction was performed in a total volume of 10 μl of 10 mM Tris-HCl, pH 8.3, 100 mM KCl and 1 mM.  MgCl_Two0.01 pmole of the obtained substrate DNA and 1 pmole of the pilot oligonucleotide It contained leotide. Incubate the reaction at 55 ° C for 30 minutes and stop Stopped by addition of l. Heat the sample at 75 ° C for 2 minutes, immediately followed by 7M urea Electrophoresis on a 10% polyacrylamide gel (19: 1 cross-link) in 0.5X TBE buffer containing I took it.   The results were visualized by autoradiography, and together with the enzymes shown below, FIG. Shown in B. I is native Taq DNAP, II is native Tfl DNAP and III is shown in FIG. 16E. Cleavedase^TMBX enzyme, IV is the cleavedase shown in FIG.^TMB B is the enzyme, V is the mutant shown in FIG. 18B, and VI is the mutant shown in FIG. 16G. Cleavease^TMBN enzyme. "Standardize" the comparison using structure 2 You. For example, to produce the same amount of cleavage in structure 2 in 30 minutes, 50 ng of Taq DNAP And 300ng of Cleavase^TMIt turned out that BN enzyme was required. These conditions Below, native Taq DNAP is unable to cleave structure 3 to a significant degree. Heaven Tfl DNAP, of course, cleaves Structure 3 in such a way as to give rise to a variety of products.   In contrast, all of the mutants tested cleave the linear duplex of structure 3. This finding indicates that this feature of mutant DNA polymerases is It shows that it matches the qualitative polymerase.                                 Example 5                          By thermostable DNAP     Five' Exonuclease hydrolytic cleavage ("Nibbling")   Thermostable DNAP (including those of the present invention) is the 5 'end of a linear double stranded nucleic acid construct. True 5 'exonuclease capable of nibbling the edges Was found to have. In this example, a 206 base pair DNA double-stranded substrate (described above) Use again). In this case, the substrate is 1 in the polymerase chain reaction. Pieces³²Generated using a P-labeled primer and one unlabeled primer. Open The cleavage reaction was performed in a total volume of 10 μl of 10 mM Tris-Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl_TwoIn, 0.01 pmol heat-denatured, end-labeled substrate DNA (with unlabeled strand), 5 pm ol pilot oligonucleotide (see pilot oligo in FIG. 13A) and 0.5 μl of the clumps in 0.5 units of DNAP Taq or E. coli extract (see above) Boase^TMIt consisted of the BB enzyme.   The reaction is started at 65 ° C with the addition of the pre-warmed enzyme and then for 30 minutes. Changed to final incubation temperature. The results are shown in FIG. 25A. Sun of lanes 1-4 Pull is the result in the presence of native Taq DNAP, lanes 5-8 are clean Bouase^TMThe results in the presence of BB are shown. Reactions in lanes 1, 2, 5, and 6 Was performed at 65 ° C., and the reactions in lanes 3, 4, 7, and 8 were performed at 50 ° C. And All reactions were 8 μl of 95% formaldehyde containing 20 mM EDTA and 0.05% marker dye. Stopped by addition of amide solution. 10 containing 7M urea dissolved in 0.5X TBE buffer Immediately prior to electrophoresis running on a% acrylamide gel (19: 1 cross-linking), Heated to 75 ° C. for 2 minutes. The expected products in reactions 1, 2, 5 and 6 are long Is 85 nucleotides. The products expected in reactions 3 and 7 are of length 27 nucleotides. Reactions 4 and 8 were performed without the addition of pilot and The length of the product should stay at 206 nucleotides. Found at 24 nucleotides The faint band is the PCR-derived residual end-labeled primer.   Cleavase under these conditions^TMIn species with very little BB enzyme, The emergence of all is a surprising result, as this enzyme completely hydrolyzes the substrate Suggests the possibility. Lanes 5-8 (antibodies performed using deletion mutants) A) To confirm the component of the fastest migrating band seen in Samples of duplex versus T7 gene 6 exonuclease (USB) or calf intestinal Treat with Lucari phosphatase (Promega) according to manufacturer's instructions, Intelligible mononucleotide (lane a in FIG. 25B) or free³²P-labeled inorganic phosphate ( Each of the lanes b) of FIG. 25B was generated. Lanes of these products and panel A 7 was dissolved in 0.5X TBE buffer, 20% acrylamide containing 7M urea. Fractionation was performed by short-time electrophoresis in Dogel (19: 1 cross-linking). Thus, Cleavure -Se^TMThe BB enzyme can convert a substrate to a mononucleotide.                                 Example 6                     Nibbling is duplex dependent   Enzyme Cleavase^TMNibbling with BB is duplex dependent. This implementation In the example, all four unlabeled dNTPs were combined using the unlabeled 206 bp fragment as a template. Α-³²15 cycles of primer extension incorporating P-labeled dCTP resulted in 20 A 6-mer internally labeled single strand was generated. Unmodified in 0.5X TBE buffer Single-stranded and double-stranded by electrophoresis in hydrophobic 6% polyacrylamide gel (29: 1 cross-linking) Chain products are fractionated, visualized by autoradiography, and excised from the gel , Eluted by passive diffusion and concentrated by ethanol precipitation.   The cleavage reaction was performed in a total volume of 40 μl of 10 Tris-Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl_TwoIn, 0.04 pmol substrate DNA and 0.2 μl enzyme cleavease^TMBB (E. coli described above) Extract). Start reaction by adding pre-warmed enzyme I let it. Take aliquots of 10 μl after 5, 10, 20 and 30 minutes, 30 mM EDTA and 0 Prepared tubes containing 8 μl of 95% formamide solution containing .05% marker dye Moved to 10% acrylamide containing 7M urea dissolved in 0.5X TBE buffer Heat the sample to 75 ° C for 2 minutes just before electrophoresis running in a gel (19: 1 cross-link). Was. As shown in FIG. 26, the results were visualized by autoradiography. joy Crab, Cleavase^TMCleavage by the BB enzyme depends on the duplex structure. 206-mer Cleavage of the duplex is complete, whereas cleavage of the single-stranded structure is not detected.                                 Example 7                         Purification of Cleve-ase ^TM enzyme   As described above, the expressed thermostable protein (ie, 5 'nuclease) Was isolated by a crude extract of bacterial cells. Next, the precipitated E. coli Was removed by centrifugation together with the cell lysate. In this embodiment, the cleaving^TMBN Cells expressing the clone were cultured and harvested (500 g). E. coli 1g each (wet weight ), 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 μM NaCl) Was added. Cells were lysed with 200 μg / ml lysozyme for 20 minutes at room temperature. Next, deoxycholic acid was added to a final concentration of 0.2% and the mixture was allowed to stand at room temperature. Incubated for 15 minutes.   Lysates were sonicated for about 6-8 minutes at 0 ° C. Centrifuge the precipitate (39,000g for 20 minutes) ). Add polyethyleneimine to the supernatant (0.5%) and mix the mixture on ice for 1 Incubated for 5 minutes.   The mixture was centrifuged (5,000 g for 15 minutes) and the supernatant was collected. 30 minutes at 60 ℃ And centrifuged again (5,000 g for 15 minutes) and the supernatant was collected again.   The supernatant was precipitated for 15 minutes at 4 ° C. using 35% ammonium sulfate. Next The mixture was centrifuged (5,000 g for 15 minutes) and the supernatant was removed. Next, the precipitate was washed with 0.25M KCl , 20 mM Tris, pH 7.6, 0.2% Tween and 0.1 EDTA. Combination buffer consists of 40 mM imidazole, 4 M NaCl, 160 mM Tris-HCl, pH 7.9) It was dialyzed as an external solution.   Next, Ni⁺⁺The solubilized protein was purified using a column (Novagen). Loose coupling Pour the impinging liquid up to the top of the column bed and allow it to drain well. Loaded in the system. A flow rate of approximately 10 column volumes per hour is optimal for efficient purification. You. If the flow rate is too high, impurities contaminating the eluted fraction will increase.   Wash the column with 25 ml (10 volumes) 1X binding buffer, then 15 ml (6 volumes) 1X wash Purification buffer (8X wash buffer is 480mM imidazole, 4M NaCl, 160mM Tris-HCl, p H7.9). 15 ml (6 volumes) of 1X elution buffer (4X elution buffer 4mM imidazole, 2M NaCl, 80mM Tris-HCl, pH7.9) To elute the bound proteins. Next, using 35% ammonium sulfate, Reprecipitate the protein as described above. Next, dissolve the precipitate and add 20 mM Tris, 100 mM KC Dialysis was performed using 1,1 mM EDTA as an external solution. 0.1% each of Tween 20 and NP-40 And stored at 4 ° C.                                 Example 8     Five' Nucleases cleave nucleic acid substrates at regions of naturally occurring secondary structure   In Example 1C (FIG. 13, lane 9), the pilot oligonucleotide 5'-nucleic acids that recognize and cleave nucleic acid substrates in the region of the naturally occurring secondary structure in the absence The ability of rease was demonstrated (ie, primer-independent cleavage). 10mM Tris-HC l, pH 8.5 and 1.5 mM MgCl_TwoA 206 bp DNA substrate (single powder) in a buffer containing DNAP Taq was incubated at 50 ° C. in the presence of an end-labeled, double-stranded template). The associated (ie, naturally occurring) structure in the DNA substrate is It was cleaved by nuclease activity. This cleavage results in three prominent fragments (FIG. 13, Lane 9) was generated. This cleavage pattern is the “fingerprint” of the DNA template. "I will provide a.   The ability of 5 'nucleases to cleave naturally occurring structures in nucleic acid templates (structure Specific cleavage) is an internalization of a nucleic acid without prior knowledge of the specific sequence of the nucleic acid. It is useful for detecting differences in local sequences. Mutation of nucleic acid using 5 'nuclease Scan for differences [eg single base changes (point mutations), small insertions or deletions, etc.] The following series of experiments were performed to develop a general method. A. MgCl in the cleavage reaction product_TwoMnCl_TwoSubstitution enhances the cleavage pattern I let you.   Cleavage pattern created by 5 'nuclease activity on double-stranded DNA substrates Effect on Mg²⁺Mn²⁺The effect of the substitution was investigated. Wild type (SEQ ID NO: 27) or Is derived from exon 4 of mutant G419R (SEQ ID NO: 28) tyrosinase gene Was prepared by PCR as follows.   A pair of primers, 5 'biotin-CACCGTCCTCTTCAAGAAG3 (SEQ ID NO: 29) and 5 'fluorescein-CTGAATCTTGTAGATAGCTA3' (SEQ ID NO: 30) PCR was started. Synthetic primers were obtained from Promega. Primer Was labeled at the 5 'end with biotin or fluorescein during synthesis.   Mutant G419R (King R.A. et al., (1991) Mol. Biol. Med. 8:19; The target DNA for the production of the 157 bp fragment of 339 bp PCR product generated using homozygous genomic DNA (SEQ ID NO: 3) 1). Genomic DNA is standardized from peripheral blood leukocytes isolated from patients. Was isolated using. This 339 bp PCR product was prepared as follows.   A symmetric PCR reaction was performed using 10 ng of genomic D from the 419 mutant in 1X PCR buffer. NA, 100 pmol of primer 5 'biotin-GCCTTATTTTACTTTAAAAAT-3' (SEQ ID NO: 32), 100 pmol of primer 5 'fluorescein-TAAAGTTTTGTG TTATCTCA-3' ( SEQ ID NO: 33) and 50 μM each of dNTPs. SEQ ID NOs: 32 and 33 Primers were obtained from Integrated DNA Technologies, Coralville, IA. Four A test tube containing 5 μl of the above mixture was overlaid with 2 drops of light mineral oil and tested. The tube was heated to 95 ° C. for 1 minute. Next, 1.25 units in 5 μl of 1 × PCR buffer Taq polymerase was added as an enzyme. Heat the test tube to 94 ° C for 40 seconds and 50 seconds While cooling to 55 ° C. and heating to 72 ° C. for 70 seconds, this was repeated 29 times. And the last After the repetition, the mixture was incubated at 72 ° C for 5 minutes.   The PCR product was gel purified as follows. Dissolve in buffer containing 0.5X TBE The product was fractionated by electrophoresis in a purified 6% polyacrylamide gel (29: 1 cross-linking). Was. Visualize the DNA by ethidium bromide staining and cut the 339 bp fragment from the gel. Started. 0.5M NH from the gel sections by overnight passive diffusion_FourOAc, 0.1% SDS And eluted in a solution containing 0.1 M EDTA. Next, 4 μg of glycogen The DNA was precipitated with ethanol in the presence of carrier. This DNA is And resuspended in 40 μl of TE (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).   Targeting in asymmetric PCR to generate a 157 bp fragment from the 419 mutant The purified 339 bp 419 PCR fragment was used. Asymmetric PCR reactions are supported by 1X PCR 100 pmol of the biotinylated primer of SEQ ID NO: 32 in the solution, 1 pmol of SEQ ID NO: 33 Fluoresceinized primers and 50 μM of each dNTP. Above 45μl The test tube containing the above mixture is overlaid with 2 drops of light mineral oil, and the test tube is kept for 5 seconds. During which time it was cooled to 70 ° C. Next, 1.25 U in 5 μl of 1X PCR buffer Taq polymerase was added as a knit enzyme. Heat the test tube to 95 ° C for 45 seconds. , Cooled to 50 ° C for 45 seconds, heated to 72 ° C for 1 minute and 15 seconds, and this was repeated 30 times. Soshi And incubated at 72 ° C. for 5 minutes after the last repetition.   The asymmetric PCR product was gel purified as follows. Buffer containing 0.5X TBE The product was analyzed by electrophoresis in a 6% polyacrylamide gel (29: 1 cross-linking) dissolved in Fractionated. DNA was visualized by ethidium bromide staining. Double-stranded DNA is The difference in mobility commonly found in single-stranded DNA generated by asymmetric PCR Better than single-stranded DNA (single-stranded and double-stranded in asymmetric PCR). Both main chain products are produced: typically single chain products are slower in the gel It has a moving velocity and appears closer to the origin than the double-stranded product). 419 mutant The corresponding double-stranded 157 bp substrate (SEQ ID NO: 28) was excised from the gel.   Above except that the target DNA was 10 ng supercoiled pcTYR-N1Tyr plasmid DNA A 157 bp wild-type fragment of the 419 mutant was generated by asymmetric PCR in the same manner as . This pcTYR-N1Tyr plasmid contains the complete wild-type tyrosinase cDNA [ Geibel, L.B., et al., (1991) Genomics 9: 435].   Following asymmetric PCR, the reaction products are separated on an acrylamide gel as described above. , Double-stranded fragments of interest were excised, eluted, and precipitated. Settling The 157 bp wild type (SEQ ID NO: 27) and 419 mutant (SEQ ID NO: 28) fragments were Resuspended in 40 μl TE.   The cleavage reaction solution was a 10 mM MOPS, pH 8.2, 1 mM divalent cation (MgCl_TwoMa Or MnCl_Two) And 100 fmol of the resulting double-stranded substrate in 1 unit of DNAP Taq DNA (the substrate contains a biotin moiety at the 5 'end of the sense strand). Anti The reaction solution was overlaid with one drop of light mineral oil. Heat the reaction solution to 95 ° C for 5 seconds to Denatured and then rapidly cooled the tubes to 65 ° C. (this step involves the complementary base By allowing the formation of inter-chain hydrogen bonds between them, DNA has a unique secondary structure It is possible to have). The above reaction is heated to 95 ° C for 5 seconds, then the temperature is increased rapidly. Thermocycler programmed to lower to 65 ° C (MJ Research, Waterow) n, MA). Alternatively, heat the test tube to 95 ° C. You can also put it in the block by hand and then transfer it to a second heat block set at 65 ° C. Wear.   The reaction was incubated at 65 ° C. for 10 minutes, and the reaction was stopped by adding 8 μl of stop buffer. Response was stopped. The sample was heated to 72 ° C. for 2 minutes. 5 μl of each reaction solution was added to 0.5X 10% polyacrylamide gel containing 7M urea dissolved in a buffer containing TBE (19 : 1 cross-linking).   After electrophoresis, remove the gel plate and leave the gel flat on one plate I did it. Pre-moistened with 0.5X TBE, with 0.2μm pores A charged nylon film (Schleicher and Schuell, Keene, NH) is exposed Placed on top of luamide gel. Remove all air bubbles trapped between the gel and the membrane Removed. Next, place 2 pieces of 3MM filter paper (Whatman) on the membrane and place it on another glass plate. , And sandwich the sandwich with binder clips. Migration (tiger Was allowed to proceed overnight. After transfer, carefully remove the membrane from the gel and air dry. Let dry. After drying completely, 1.2X Sequenase Images blocking buffer (Un The membrane was washed for 30 minutes in ited States Biochemical). Membrane cm^TwoPer buffer 3 / 10 ml was used. Streptavidin-alkaline phosphatase conjugate (SAAP, Unite d States Biochemical) in the blocking solution above until a dilution of 1: 4000. Were added immediately and stirred for 15 minutes. Rinse the membrane briefly with water, then 0.1% sodium dodecyl sulfate In 1X SAAP buffer (100mM Tris-HCl, pH10; 50mM NaCl) containing lium (SDS) Washed 3 times (5 min / 1 wash). 0.5ml buffer / cm^TwoBuffer between each wash. Rinse with water. Similarly, for fluorescein-labeled DNA, anti-fluorescein 1: 20,000 in fragments (Boehringer Mannheim Biochemicals, Indianapolis, IN) Add at final dilution, then 1X SAAP buffer containing 0.1% SDS and 0.025% Tween20 Can be washed three times in the buffer (5 minutes / wash). Next, the membrane was made 1X S without SDS. Wash once in AAP buffer, completely drain and heat seal plastic I put it in a bag. Using a sterile pipette tip, 0.05 ml / cm^TwoCDP-Star^TM(Tropi x, Bedford, MA) was added to the bag and allowed to spread over the membrane for 5 minutes. film From which all excess liquid and air bubbles were removed. Next, use X-ray film (Kodax XRP) For the first 30 minutes. Adjust exposure time if needed for fractionation and clarity did. The results are shown in FIG.   In FIG. 27, the lane marked “M” contains the molecular weight marker . The marker fragment was digested with pUC19 using HaeIII, followed by terminal transfer. A biotinylated dideoxynucleotide (Boehri nger Mannheim Biochemicals, Indianapolis, Ind.). lane 1, 3 and 5 show MgCl in the absence of DNAP Taq enzyme (lane 1)._TwoAnd yeast In the presence of nitrogen (lane 3) or MnCl_TwoAnd in the presence of enzyme (lane 5) Contains reaction products from incubation of wild-type 157 nucleotide substrates You. Lanes 2, 4 and 6 show MgCl 2 in the absence of enzyme (lane 2)._TwoAnd enzymes (Lane 4) or MnCl_TwoAnd 4 in the presence of enzyme (lane 6) Reaction generation by incubation of 157 nucleotide substrates from 19 mutants Material.   FIG. 27 shows MgCl in the cleavage reaction._TwoRather than MnCl_TwoCleavage with increased use of This indicates that a pattern is generated. All of the cleavage products are at one end of the gel It is desirable that the cleavage products be of different sizes so that they do not clump. Cleavage The ability to disperse the product over the entire length of the gel depends on the wild-type and mutant substrates. It is more likely that changes in the cleavage products between will be identified. FIG. Mg as divalent cation²⁺If used, most of the cleavage products will be at the top of the gel. It shows that they flock. In contrast, Mn as a divalent cation²⁺Where was used In some cases, the substrate also has a structure that, when cleaved, results in a product with significantly different mobility. One. These results indicate that Mn²⁺Is a preferred divalent cation Are shown. B. 5 'nuclease cleavage of different DNAs of similar size creates unique cleavage fragments Generate.   4 cleaved DNA substrates of similar size^TMA with BN enzyme By incubating, a specific DNA fragment has a unique cleavage pattern or The ability of the 5 'nuclease to generate a "fingerprint" was demonstrated. used The four DNA substrates are the sense (or exon 4) of the wild-type tyrosinase gene. Code) chain-derived 157 nucleotide fragment (SEQ ID NO: 34); wild-type tyrosinase 157 nucleotides from the antisense (or non-coding) strand of exon 4 of the gene Fragment (SEQ ID NO: 35); a 165 nucleotide DNA fragment derived from pGEM3Zf (+) (sequence No. 36); and a 206 nucleotide DNA fragment derived from pGEM3Zf (+) (SEQ ID NO: 3) 7). These DNA substrates have biotin or fluorescein at the 5 'or 3' end. It contained a CEJN label. These substrates were made as follows.   Sense and antisense corresponding to exon 4 of wild-type tyrosinase gene Double-stranded 157 nucleotides in length using symmetric PCR to generate single-stranded substrates A DNA fragment (SEQ ID NO: 27) was generated. Target of symmetric PCR is wild-type tyrosin It was a genomic DNA containing the ze gene. The symmetric PCR reaction is 50 μl of 1X PCR buffer. 50-100 ng of wild-type genomic DNA, 25 pmol of each primer (SEQ ID NO: 42 and 43), from 50 μM each of dNTPs and 1.25 units of Taq polymerase Had become. The reaction mixture is overlaid with 2 drops of light mineral oil and the test tube is kept for 30 seconds. Heat to 94 ° C, cool to 50 ° C for 1 minute, heat to 72 ° C for 2 minutes, repeat this 30 times Was. The double-stranded PCR product is gel purified and precipitated as described in a) above, Resuspended in 40 μl TE buffer.   Using the above 157 bp wild type DNA fragment (SEQ ID NO: 27), two asymmetric PCs The single-stranded sense and antisense 157 nucleotide DNA fragments were Generated. The sense strand fragment targets 5 μl of the purified 157 bp fragment (SEQ ID NO: 27) And generated by asymmetric PCR. The reaction mixture for asymmetric PCR is 100 pmol of biotin-labeled sense primer to initiate reaction (SEQ ID NO: 29) And 1 pmol of fluorescein-labeled antisense primer (SEQ ID NO: 30) Except where used, it was the same as described for asymmetric PCR. Antisen Using the 5 μl of the above purified 157 bp fragment as a target by asymmetric PCR. Generated. The reaction conditions for asymmetric PCR were such that 1 pmol of sense probe was required to initiate the reaction. Primer (SEQ ID NO: 29) and 100 pmol of antisense primer (SEQ ID NO: 30) Same as described above for symmetric PCR except that 30) was used Met.   The reaction conditions for asymmetric PCR are 95 ° C for 45 seconds, 50 ° C for 45 seconds, 72 ° C for 1 minute and 15 seconds 30 times. Repeat and incubate at 72 ° C for 5 minutes after the last repeat. Was. The reaction products were visualized, extracted and collected as described above. Single-stranded DNA , As compared to the control double-stranded DNA.   The single-stranded 165 nucleotide fragment (SEQ ID NO: 36) derived from pGEM3Zf (+) was Generated by R. The PCR reaction was performed with 50 pmol of 5 'biotin in 1X PCR buffer. -AGCGGATAACAATTTCACACAGGA-3 ′ (SEQ ID NO: 38: Promega) and 1 pmol CACGG ATCCTAATACGACTCACTATAGGG-3 '(SEQ ID NO: 39: Integrated DNA Technologies, C oralville, IA) and 50 μM each of dNTPs. 45 μl of the above reaction mixture Overlay 2 drops of light mineral oil, heat test tube to 95 ° C for 5 seconds, cool to 70 ° C did. Next, Taq polyether was used as 1.25 units of enzyme in 5 μl of 1X PCR buffer. Merase was added. Heat test tube to 95 ° C for 45 seconds, cool to 50 ° C for 45 seconds, 1 minute Heated to 72 ° C. for 15 seconds, this was repeated 30 times. And after the last iteration, 72 Incubated at 5 ° C for 5 minutes. Visualize and extract the reaction products as described above, Collected. The 164 nucleotide DNA fragment has a higher mobility than the control double-stranded DNA. Identified by the difference between   A 206 nucleotide DNA fragment (SEQ ID NO: 37) was converted to 1 pmol of a double-stranded 206 bp PC R product (produced as described in Example 1C) and 50 pmol of primer Using 5'-CGCCAGGGTTTTCCCAGTCACGAC-3 (SEQ ID NO: 40), the procedure was performed as described above. Prepared by asymmetric PCR. Heat the test tube to 95 ° C for 45 seconds and cool to 63 ° C for 45 seconds And heated to 72 ° C. for 1 minute and 15 seconds, and this was repeated 15 times. After the last iteration, 7 Incubated at 2 ° C for 5 minutes. Visualize and extract the reaction products as described above, Collected. The 206 nucleotide DNA fragment has a higher mobility than the control double-stranded DNA. Identified by the difference between The precipitated DNA was resuspended in 70 μl of TE buffer.   10-20 units of terminal deoxynucleotidyl tra in 50 μl reaction Using the transferase (TdT) (Promega), 25 μl of the 3 ′ end of the above product was vialized. Otinized. The reaction solution was 0.5 mmol of biotin-16-ddUTP (Boehringer Mannhei m) and 1X TdT buffer (500 mM cacodylate buffer, pH 6.8, 5 mM CoCl_Two, 0.5mM D TT and 500 μg / ml BSA). Incubate tubes at 37 ° C for 15 minutes Then, ethanol precipitation was performed in the presence of 4 μg of glycogen. DNA Ethanol precipitated again and then resuspended in 25 μl TE.   The cleavage reaction was performed with approximately 100 fmol of substrate DNA and 250 ng of enzyme cleavase.^TMBN Using 1 mM MnCl_TwoIn a final volume of 10 μl of 10 mM MOPS, pH 8.2. Was. Enzyme Cleavase^TMParallel reactions lacking BN (no control enzyme) 1/3 DNA template (about 33 fmol Except that the respective molds were used.   Each substrate DNA was prepared using 2 mM MnCl_TwoMixed with 5 μl of 10 mM MOPS, pH 8.2 containing Placed in 00 μl thin-walled microcentrifuge tubes (BioRad, Hercules, CA). 2 drops in this solution Of light mineral oil. Heat the tube to 95 ° C for 5 seconds to denature the substrate. The tube was then rapidly cooled to 65 ° C.   5 μl of MnCl_Two1 μl of enzyme cleavease in 10 mM MOPS, pH 8.2^TM BN [1X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KC 1, 10 mg / ml BSA) 250 ng / μl] The cleavage reaction was immediately started. Allow the enzyme solution to come to room temperature before adding to the cleavage reaction. I left it. After 5 minutes at 65 ° C., the reaction was stopped by adding 8 μl of stop buffer. . The sample was heated to 72 ° C for 2 minutes and 5 μl of each reaction was added to a buffer containing 0.5X TBE. Electrophoresis in 10% polyacrylamide gel containing 7M urea (19: 1 cross-link) dissolved in buffer By fractionation.   After electrophoresis, the gel plate was removed and positively charged with 0.45 μm pores A nylon membrane (United States Biochemical) was applied. DNA as described above And dry the membrane and wash in 1.2X Sequenase Images blocking buffer. It was cleaned and treated with SAAP buffer. CDP-Star^TMInstead of Lumiphos-530 (United Stat es Biochemical) or Quantum Yield Chemiluminescent Substrate (Promega) Generated. The film was then exposed to an X-ray film as described above. Auto obtained The radiograph is shown in FIG.   Fig. 28 shows the Cleavase^TMIn the presence or absence of the BN enzyme, the above four Shows the results of incubation of the substrates. Four sets of reactions are shown. Set Toe 1 is Cleavease^TMTyrosiners in the absence or presence of BN enzyme Incubation of a 157 nucleotide sense strand fragment of the ze gene (SEQ ID NO: 34) It contains the reaction product of the origin. Set 2 is Cleavease^TMAbsence of BN enzyme 157 nucleotide antisense strand of tyrosinase gene under or in the presence Contains the reaction product from incubation of the fragment (SEQ ID NO: 35). Set To 3 is Cleavease^TMPlasmid in absence or presence of BN enzyme Incubation of 165 base bottom chain fragment (SEQ ID NO: 36) of pGEM3Zf (+) It contains the reaction product derived from the solution. Set 4 is Cleavease^TMNon-BN enzyme 206 base top strand fragment of plasmid pGEM3Zf (+) in the presence or absence Contains the reaction product from the incubation of (SEQ ID NO: 37). "M" Marked lanes contain biotin-labeled molecular weight markers prepared as described above. I do. Cleavease^TMNo substrate cleavage is seen in the absence of the BN enzyme . Cleavease^TMIn the presence of the BN enzyme, each substrate is cleaved and a set of unique Yields a cleavage product. Fractionation of these cleavage products on polyacrylamide gels You will see a unique pattern or fingerprint for each substrate DNA. It is. Thus, while the four substrates are similar in size (157-206 bases) , Cleavase^TMBN enzyme yields a unique set of cleavage products from each substrate You. These unique cleavage patterns indicate the characteristic conformation of each substrate DNA. Originated from   The present invention creates unique cleavage patterns for two or more DNA substrates of the same size. Are considered as part of the method of detecting gene mutations. this The method uses a normal (or wild-type or non-mutated) substrate It is compared with a substrate from a patient suspected of having a mutation in quality. This place If so, the two substrates will be the same length. And found in the wild-type control substrate Scrutinize patient DNA substrates for conformational changes compared to patterns To do so, a cleavage reaction will be used.                                 Example 9           The cleavage elicited by the Cleavase ^™ BN enzyme is               Detects single base changes in DNA substrates.   DNAs of multiple DNA substrates of the same size, but with a single base change between them Cleavase that cleaves substrates^TMThe ability of the BN enzyme is shown here. As a model system The human tyrosinase gene was selected. Because the exon 4 of this gene Many point mutations have been identified [Spritz, R.A. (1994) Human Mol ecular Genetics 3: 1469]. Mutations in the tyrosinase gene are Causes albinism of the skin.   Three single-stranded substrate DNAs were prepared. These substrates have a biotin label at the 5 'end. contains. The wild-type substrate is 157 nucleotides derived from the sense strand of the human tyrosinase gene. Contains an otide fragment [(SEQ ID NO: 34); Geibel, L.B. et al., (1991) Genomics 9: 435]. I do. Two substrates with mutations were used. The 419 substrate (SEQ ID NO: 41) is Tyrosinase projections having glycine (GGA) to arginine (AGA) substitutions Derived from mutant G418R. This mutant is at nucleotide 2675 It differs from the wild type exon 4 fragment by one base change [King, RA et al., (19) 91) Mol. Biol. Med. 8:19]. The 422 substrate (SEQ ID NO: 42) is arginine (CGG) Tyrosinase mutant R422 having a substitution of glutamine (CAG) for Derived from Q. This mutant has a single base change at nucleotide 2685. Are more distinct from the wild-type exon 4 fragment [Geibel, L.B. et al., (1991) J. Am. Clin.Inv est. 87: 1119].   A single-stranded DNA having a biotin label at the 5 'end for each substrate is described in Example 8a. Was generated using asymmetric PCR. However, not a double-stranded product, but a single-stranded PC The R product was recovered from the gel.   Each DNA was amplified using the following primer pairs (419 and 422 mutants Are located inside the exon 4 fragment amplified by this primer pair. did Thus, identical primer pairs to amplify the wild-type and two mutant templates Can be used). The primer set forth in SEQ ID NO: 29 (sense primer ) Contains a biotin label at the 5 ′ end and is used as an antisense primer of SEQ ID NO: 30. It was used in a 100-fold excess.   The following template was used to generate a single-stranded substrate. 10 ng supercoiled plasmid DNA was converted to wild type (plasmid pcTYR-N1Tyr) or 422 mutant (plasmid p cTYR-A422) was used as a target to generate a 157 nucleotide fragment. 419 suddenly 5 μl of genome derived from genomic DNA homologous to the mutant (described in Example 8a) Of the purified 339 bp PCR fragment (SEQ ID NO: 31) Piece (SEQ ID NO: 41) was used as a target to generate.   For each of the target DNAs, the asymmetric PCR reaction was performed at 100p in 1X PCR buffer. consisting of 1 mol of SEQ ID NO: 29 and 1 pmol of SEQ ID NO: 30, and 50 μM of each dNTP Was. The reaction mixture (45 μl) was overlaid with 2 drops of light mineral oil and the test tube was Heat to 5 ° C and cool to 70 ° C. Next, 1.25 units in 5 μl of 1X PCR buffer Taq polymerase was added as a kit enzyme. Heat the test tube to 95 ° C for 45 seconds, It was cooled to 50 ° C. for 5 seconds and heated to 72 ° C. for 1 minute and 15 seconds, and this was repeated 30 times. And After the last repeat, incubated at 72 ° C. for 5 minutes. Single-stranded PCR product Was gel purified as above, precipitated and resuspended in 40 μl of TE buffer.   The cleavage reaction was performed as described in Example 8b. Allow sample to 72 ° C for 2 minutes Heat and add 7 μl of each reaction to 10% 7M urea in buffer containing 0.5X TBE Fractionation was performed by electrophoresis in polyacrylamide gel (19: 1 cross-linking). After electrophoresis, The DNA was transferred to a nylon membrane and subjected to SAAP and CDPStar as described in Example 8a. Processed. FIG. 29 shows the obtained autoradiograph.   In FIG. 29, lanes marked “M” were prepared as described in Example 8. Containing a molecular weight marker. Lanes 1-3 are wild type (SEQ ID NO: 34), 41 9 mutants (SEQ ID NO: 41) and 422 mutants (SEQ ID NO: 42), respectively No control enzyme. Lane 4 contains cleavage product from wild-type template I do. Lane 5 contains cleavage products from the 419 mutant. Lane 6 has 422 bumps It contains cleavage products from the mutant.   Figure 29 shows Cleavase^TMBy digestion of the three template DNAs with the BN enzyme , Indicating that a similar but distinctly different pattern of cleavage products is produced I have. Several new bands of 53 nucleotides were found in the digest of mutant 422. It appears in the vicinity, but when compared with the wild type in the digest of mutant 419 about Note that there is no band below 40 nucleotides.   The three template DNAs differ only in one of the 157 nucleotides. However, a unique cleavage fragment pattern was generated for each. Therefore, 15 One base change in the 7 nucleotide fragment is cleaved^TMApproved by enzymes This leads to different secondary structures that are perceived.                                 Example 10                     By the enzyme Cleavase ^™ BN             One base change detected in large DNA fragment   The previous example shows that^TMUsing the 5 'nuclease activity of the BN enzyme, The ability to detect one point mutation in a nucleotide DNA fragment showed that. In this example, one point mutation is detected in a larger DNA fragment. Cleavease^TM2 shows the ability of the BN enzyme.   Fragments from the 422 tyrosinase mutant, which were gradually increased in size, were And compared to fragments of the same size from the type tyrosinase gene. 4 sets of single-chain groups Quality was used. That is, 1) wild type (SEQ ID NO: 34) and 422 mutant ( 157 nucleotide template from the sense strand of exon 4 of SEQ ID NO: 42); Exons from the native (SEQ ID NO: 43) and 422 mutant (SEQ ID NO: 44) 378 nucleotide fragment containing 4 and 5; 3) wild type (SEQ ID NO: 45) and 1.059k containing exons 1-4 from the 422 mutant (SEQ ID NO: 46) b fragment; and 4) wild-type (SEQ ID NO: 47) and 422 mutant (SEQ ID NO: 48) This is a 1.587 kb fragment containing exons 1 to 5 derived from. Wild-type template and 42 The only difference between the two mutant templates is that exon, regardless of template length used 4 from G to A. The point mutation from G to A is 157 bases, 378 From the labeled ends of the base, 1.059 kb and 1.6 kb substrate DNA, 27, 27, respectively Located at 929 and 1237 nucleotides. A) Preparation of substrate DNA   In the PCR for generating the above substrate DNA, wild-type [pcTYR-N1Tyr, B ouchard, B. et al. Exp. Med. 169: 2029] or 422 mutant [pcTY R-A422, Giebel, L.b. et al., (1991) 87: 1119] A CD containing the tyrosinase gene. The NA clone was used as the target DNA. A primer consisting of SEQ ID NOs: 42 and 43 157 bp Dp from mutant or wild-type cDNA clones An NA fragment was generated. Using a primer pair consisting of SEQ ID NOs: 29 and 49, Generated double-stranded 378 bp DNA fragment from mutant or wild-type cDNA clone . Mutant or wild-type using a primer pair consisting of SEQ ID NOs: 50 and 30 A double-stranded 1.059 kbp DNA fragment derived from the cDNA clone was generated. SEQ ID NO: 51 and Mutant or wild-type cDNA clones using a primer pair consisting of A double-stranded 1.587 kbp DNA fragment of origin was generated. In each case, the sense strand The immer contained a biotin label at the 5 'end.   The PCR reaction was performed as follows. 50 mM of each dNTP and 1 in 1X PCR buffer In a 100 μl reaction containing μM of each primer of the specific primer pair, 1 ~ 2ng of wild type or 422 mutant derived plasmid DNA as target DNA Using. A test tube containing the above mixture was overlaid with 3 drops of light mineral oil, Heated to 94 ° C for a minute and then cooled to 70 ° C. Next, 2.5 uL in 5 μl of 1X PCR buffer Taq polymerase was added as a knit enzyme. Heat the test tube to 93 ° C for 45 seconds. Cooled to 52 ° C. for 2 minutes, heated to 72 ° C. for 1 minute and 45 seconds, and repeated 35 times. Soshi And incubated at 72 ° C. for 5 minutes after the last repetition.   After the PCR reaction, a QIA Quick-Spin PCR purification kit (Qiagen, Inc. Chatsworth, C A) was used according to the manufacturer's instructions to remove excess primer. DNA Eluted in 50 μl of TE. Of each double-stranded fragment from the wild-type and 422 mutant genes The sense strand was isolated as follows. Streptavidin-coated paramagnetic beads (Dynal  M280 beads) [0.5 mg in 50 μl; 2X binding and washing (B & W) buffer (2 M NaCl, 10 mM Tr is-HCl, pH 7.5, 1 mM EDTA, 0.1% Tween 20). Added to each of the CR products. Occasionally shake the sample at room temperature. Incubated for 15 minutes. Remove the supernatant from the supernatant by exposing the test tube to a magnetic plate The beads were removed. The bead-DNA complex was washed twice in 2X B & W buffer. 100 Add μl 0.1 M NaOH to the beads and incubate the sample at room temperature for 15 minutes. Cubbed (for 157 and 378 bp DNA). For DNA fragments larger than 1kb The beads were then incubated at 47 ° C. for 30 minutes. Finally, beads-ssDN The A-complex was resuspended in 50 μl of 2 × B & W buffer and stored at 4 ° C. B) Conditions for the cleavage reaction   The cleavage reaction was performed directly on the single-stranded DNA-bead complex. 5-10 μl of DN The A-bead complex (about 100 fmol DNA) was placed in a 200 μl microcentrifuge tube and 10 μl Washed once with sterile water. Next, 1.3 mM MnCl_Two(To a final concentration of 1 mM) 7.5 μl of 10 mM MOPS, pH 8.2 was added to each test tube. Reaction test tubes must be 2 Warmed to 65 ° C for minutes. And 2.5 μl of enzyme Cleavase^TMBN (1X dilution) Cleavage was initiated by the addition of 10-50 ng) in buffer. Run the reaction at 65 ° C for 5 minutes Was.   Immediately after this 5 minute incubation, the beads were allowed to settle to the bottom of the tube and The Qing was removed and discarded. Next, 10-40 μl of stop solution was added to the beads and the sample was Incubated at 90 ° C. for 10 minutes. Fluamide / EDTA solution from beads Release the activated DNA. The beads were allowed to settle to the bottom of the test tube. Cleavage products The supernatant containing was recovered. Dissolve 2-8 μl of the supernatant solution in 0.5X TBE containing buffer 6% polyacrylamide gel containing 7M urea (19: 1 cross-linking).   After electrophoresis, the DNA was transferred to a nylon membrane and treated as described in Example 8a. Processing was performed using AP and CDPStar. FIG. 30 shows the obtained autoradiograph.   In FIG. 30, lanes marked “M” were prepared as described in Example 8. Containing a molecular weight marker. Lanes 1, 3, 5, and 7 are wild-type tyrosine. 157, 378, 1056 or 1587 nucleotide sense strand fragments from Each contains a cleavage product. Lanes 2, 4, 6, and 8 show 422 mutant 157, 378, 1056 or 1587 nucleotide sense strand fragment from tyrosinase gene Respectively.   As shown in FIG. 30, the clear cleavage seen between the wild type and the 422 mutant Pattern is unclear even when there is one base change in longer DNA fragment Did not become. Therefore, the cleavage reaction of the present invention scans large fragments of DNA. And can be used to detect mutations. Fragments longer than about 500 bp Existing methods, such as SSCP, DGGE analysis, cannot scan.                                 Example 11       Cleve ase ^TM reaction is insensitive to large changes in the reaction conditions   The above result is^TMBN enzyme is structure-specific but sequence dependent It has been shown that DNA templates can be used to probe DNA templates in a manner that does not. this The results show a conformational change in the nucleic acid caused by the sequence mutation. Cleavease is an efficient way to identify^TMThat BN enzyme can be used Indicated. This scans the nucleic acid template for sequence changes compared to the wild-type template Cleavase to develop the way^TMUses 5 'nuclease activity of BN enzyme Suggested that you can. The following example has shown that this is correct. In addition, the book The method of the invention is relatively insensitive to large changes in conditions, so this method is The following shows that it is suitable for actual battles in Bo.   First, MnCl_TwoThe effect of the change in concentration on the cleavage reaction was confirmed. Second, different The effect of different amounts of salt (KCl) on the cleavage pattern was investigated. Third, when complete cleavage In order to investigate whether or not the was obtained, a change with time was examined. Fourth, the change in temperature is a Temperature titration was performed to confirm the effect on the temperature. Next, in the enzyme concentration The enzyme was titrated to see the effect of the 50-fold change on the cleavage reaction. these The result of the experiment was Cleavase^TMThe reaction is extremely resistant to large changes in conditions It was shown. A) MnCl_TwoTitration   MnCl_TwoTo check the sensitivity of the cleavage reaction to changes in concentration, Bouase^TMIn the presence of BN enzyme (250 ng), 10 mM MOPS, pH 8.2 and various amounts of MnC l_TwoOne template was incubated in a buffer containing The cleavage reaction is as follows It was carried out as follows. 157 nucleotide sense strand fragment of tyrosinase gene (SEQ ID NO: 42; prepared by asymmetric PCR as described in Example 9) (Final concentration 0.1, 0.4, 6.8 or 10 mM MnCl_Two0.2, 4.8, 12 or Is 20mM MnCl_TwoMix with 5 μl of 10 mM MOPS, pH 8.2 containing 200 μl of wall thinner Into a microcentrifuge tube (BioRad). 10MnCl_TwoTo 10 mM MOPS, pH 8.2 (5 μl) Prepare a test tube containing 100 fmol of the mixed template DNA, and prepare enzyme-free (or uncut) No) used as control. Each reaction mixture was overlaid with one drop of light mineral oil. The test tube was heated to 95 ° C for 5 seconds and then cooled to 65 ° C.   The cleavage reaction was started and stopped as described in Example 8b. Add stop solution Thereafter, the samples were heated to 72 ° C. for 2 minutes and 8 μl of each reaction contained 0.5 × TBE Electrophoresis in 10% polyacrylamide gel containing 7M urea (19: 1 cross-link) dissolved in buffer Fractionation was performed by motion. After electrophoresis, the DNA was transferred to a nylon membrane and described in Example 8a. Treated with SAAP and CDPStar as described. Figure of the obtained autoradiograph 31.   In FIG. 31, the lane marked "M" contains the molecular weight marker. Les Lane 1 contains the no enzyme control and indicates the migration of uncleaved template DNA. Lanes 2-8 are cleaved^TMIn the presence of the BN enzyme, 10, 8, 6, 4, 2, 1 or 0 mM MnCl_TwoReaction products incubated in a buffer containing Contains a product.   FIG. 31 shows that no cleavage occurs in the absence of divalent cations. (Lane 8, 0 mM MnCl_Two). MnCl_TwoInclusion facilitates efficient generation of cleavage fragments Was done. The clearest cleavage pattern is 1 mM MnCl_Two(Lane 7) , Mn^2-Even when the concentration changed from 1 to 4 mM, little change was observed in the pattern. MnCl_TwoHigher concentrations tend to inhibit the cleavage reaction (concentrations above 6 mM). these The result is that while the cleavage reaction requires a divalent cation, the amount of divalent cation present The change has little effect on the cleavage pattern. B) Effect of salt concentration on cleavage reaction   To confirm the effect of salt concentration on the cleavage reaction, a fixed amount of^TMB 10 mM MOPS, pH 8.2, 1 mM MnCl in the presence of N enzyme (250 ng)_TwoAnd various amounts of KCl One template was incubated in a buffer containing Tyrosinase inheritance 157 base fragment (SEQ ID NO: 34; described in Example 8a) 100 fmol) in 10 mM MOPS, pH 8.2 and 1 mM MnCl_TwoContaining mild The suspension was mixed and placed in a 200 μl thin-walled microcentrifuge tube (BioRad). KCl to final concentration Was 0, 10, 20, 30, 40 or 50 mM. Final reaction volume is 10 μl there were.   10 mM MOPS, pH 8.2, 1 mM MnCl_TwoContains 33 fmol template DNA and 50 mM KCl Test tubes were prepared and used as no enzyme (or uncleaved) controls. 1 for each reaction mixture Drops of light mineral oil were overlaid. Heat the test tube to 95 ° C for 5 seconds, then to 65 ° C Cool.   The cleavage reaction was started and stopped as described in Example 8b. Add stop solution Thereafter, the samples were heated to 72 ° C. for 2 minutes and 8 μl of each reaction contained 0.5 × TBE Electrophoresis in 10% polyacrylamide gel containing 7M urea (19: 1 cross-link) dissolved in buffer Fractionation was performed by motion. After electrophoresis, the DNA was transferred to a nylon membrane and described in Example 8b. SAAP and Lumiphos-530 (United States Biochemical) or Quant as described um Yield Chemiluminescent Substrate (Promega Corp., Madison WI) Exposure. FIG. 32 shows the obtained autoradiograph.   In FIG. 32, the lane marked “M” contains the molecular weight marker. Les Lane 1 contains the no enzyme control and indicates the migration of uncleaved template DNA. Lanes 2-7 are cleaved^TM50, 40, 30, 20, 10 or more in the presence of BN enzyme Or the reaction products incubated in buffers containing 0 mM KCl, respectively. contains.   The results shown in FIG.^TMThe reaction is relatively insensitive to changes in salt concentration It indicates that it is sensitive. Cleavease^TM157 nuclei using BN enzyme When the otide tyrosinase DNA template (SEQ ID NO: 34) was incubated, KC The same cleavage pattern was obtained irrespective of the change in l concentration from 0 to 50 mM. C) Temporal change of cleavage reaction   To check how quickly the cleavage reaction is completed, a single template is Leavease^TMIncubation was performed for various lengths of time in the presence of the BN enzyme. 10m M MOPS, pH8.2, 2mM MnCl_TwoAnd 440 fmol of tyrosinase gene exon 4 157 base fragment from the sense strand (SEQ ID NO: 34; prepared as described in Example 8b) ) Was prepared consisting of a 20 μl solution containing Each time to measure Aliquots of 5 μl were placed into 200 μl thin-walled microcentrifuge tubes (BioRad) for use. Unfermented A control tube was prepared. This reaction solution was 1 mM MnCl_TwoContaining 10 mM MOPS, pH 8.2 ( The final reaction volume (10 μl) contained 33 fmol of template DNA. One drop of light for each reaction Mineral oil was overlaid. Heat the test tube to 95 ° C for 5 seconds to denature the mold, Then cooled to 65 ° C.   The cleavage reaction was initiated by the addition of the diluted enzyme mixture as described in Example 8b. Was. The reaction was stopped immediately at the indicated times by the addition of 8 μl of stop solution. Nothing Enzyme controls were incubated at 65 ° C for 10 minutes and 8 μl in the same manner as the other reactions. Was added to stop the reaction. Heat the sample to 72 ° C for 2 minutes and allow each reaction 5 μl of the solution was dissolved in a buffer containing 0.5 × TBE and 10% polyacrylic acid containing 7M urea. Fractionation was performed by electrophoresis in an amide gel (19: 1 crosslinking). After electrophoresis, remove DNA Lumiphos-530 (U) after transfer to membrane and treatment with SAAP as described in Example 8b. nited States Biochemical) or Quantum Yield chemiluminescent substrate (Promega Corp., (Madison WI) and exposed to X-ray film. The obtained autoradiograph FIG.   In FIG. 33, lanes marked “M” were prepared as described in Example 8. Containing a molecular weight marker. Lane 1 shows the enzyme-free pair incubated for 10 minutes. Contains light. Lanes 2-5 are incubated at 65 ° C for 0.1, 1, 5 or 10 minutes. It contains cleavage products from the reaction mixture. Figure 36 shows Cleavase^TMBN yeast The cleavage reaction mediated by nitrogen has been shown to be very rapid. Less than 6 seconds ( Cleavage is already seen at <0.1 min) and is complete within 1 min. These results also It shows that the same cleavage pattern is generated when the reaction is performed for 1 minute or 10 minutes. doing. D) Temperature titration of Cleavase reaction   To confirm the effect of temperature change on the cleavage pattern, the tyrosinase gene A 157-base fragment (SEQ ID NO: 34) derived from the sense strand of exon 4 Aase^TMIncubated for 5 minutes at various temperatures in the presence of BN enzyme. 100fmo of substrate DNA (prepared as described in Example 8b) was added to 2 mM MnCl_TwoContains Mix with 5 μl of 10 mM MOPS, pH 8.2 and add 200 μl of thin-walled microcentrifuge tube (BioRad) Put in. Two "no enzyme" test control tubes were prepared as described above. However, These reactions were 1 mM MnCl_Two33 fmol of substrate DNA in 10 μl of the above buffer containing Was included. One drop of light mineral oil was overlaid on each reaction solution. Test tube for 5 seconds The mold was denatured by heating to 95 ° C in a tube and then cooled to the desired temperature.   Cleavage reaction started immediately by addition of diluted enzyme mixture as described in Example 8b I let it. The tubes were brought to 55, 60, 65, 70, 75 or 80 ° C. After 5 minutes at the specified temperature The reaction was stopped by the addition of 8 μl of stop buffer.   The sample was heated to 72 ° C for 2 minutes and 5 μl of each reaction was added to a buffer containing 0.5X TBE. Electrophoresis in 10% polyacrylamide gel containing 7M urea (19: 1 cross-link) dissolved in buffer By fractionation. After electrophoresis, the DNA was transferred to a nylon membrane and described in Example 8b. Lumiphos-530 (United States Biochemical) or Q uantum Yield chemiluminescent substrate (Promega Corp., Madison WI) Lum was exposed. FIG. 34 shows the obtained autoradiograph.   In FIG. 34, lanes marked “M” were prepared as described in Example 8. Containing a molecular weight marker. Lanes 1 and 2 are at 55 ° C and 80 ° C, respectively. Contains the incubated no-enzyme control. Lanes 3-8 are 55, 60, 65, 70 Cleavase, incubated at 75 or 80 ° C, respectively^TMContains BN enzyme Contains cleavage products from the reaction solution.   Fig. 34 shows the Cleavase^TMThe reaction can be performed over a wide temperature range Is shown. In response to incubation temperatures ranging from 55 to 75 ° C, the cleavage pattern The tone gradually changed. Some bands are more sensitive to incubation at higher temperatures. Only revealed when. Probably some involved in cleavage at intermediate temperatures Would not have been advantageous at lower temperatures. As expected At the upper end of the temperature range tested, the structure was melted, and the cleavage was gradually reduced. It was becoming. At 80 ° C, cleavage is completely suppressed, presumably due to complete denaturation of the template. Re Was.   These results indicate that the cleavage reaction can be performed over a wide temperature range. ing. The ability to carry out the cleavage reaction at elevated temperatures is important. Strong mold ( In other words, if it has a (stable) secondary structure, one nucleotide change Or it is unlikely to significantly alter the cleavage pattern it produces. In an array Maximize the effect of small changes, which manifest as changes in the cleavage pattern within the target template As described above, it is possible to bring the structure to the brink of instability using high temperatures. As a result, a cleavage reaction can occur at that point. E) Cleavease^TMTitration of BN enzyme   Cleavase at various concentrations in the cleavage reaction^TMThe effect of the BN enzyme was examined. H 157 base fragment from the sense strand of exon 4 of rosinase gene (SEQ ID NO: 34) 1 00fmol to 2mM MnCl_TwoMixed with 5 μl of 10 mM MOPS, pH 8.2 containing Placed in a centrifuge tube. An enzyme-free control tube was prepared. The reaction solution was 1 mM MnCl_TwoContains It contained 33 fmol of substrate DNA in 10 μl of 10 mM MOPS, pH 8.2. Each reaction solution Was overlaid with one drop of light mineral oil. Heat the test tube to 95 ° C for 5 seconds to mold Denatured and then cooled to 65 ° C.   10, 50, 100 or 250 ng of enzyme is MnCl_TwoIn 5 μl of 10 mM MOPS, pH 8.2 1 μl of Cleavase in 1 × dilution buffer so that it is in the test tube^{T M} The cleavage reaction is immediately opened by adding a dilute enzyme mixture consisting of BN enzyme. Started. After 5 minutes of reaction at 65 ° C., the reaction was stopped by adding 8 μl of stop buffer. I let you. The sample was heated to 72 ° C. for 2 minutes and 7 μl of each reaction contained 0.5 × TBE In a 10% polyacrylamide gel containing 7M urea (19: 1 cross-linking) dissolved in buffer Fractionated by electrophoresis.   After electrophoresis, the DNA was transferred to a nylon membrane and subjected to SAAP as described in Example 8b. Lumiphos-530 (United States Biochemical) or Quantum Yield Reacted with chemiluminescent substrate (Promega Corp., Madison WI) and exposed to X-ray film . The obtained autoradiograph is shown in FIG.   In FIG. 35, the lane marked "M" contains the molecular weight marker. Les Lane 1 contains the no enzyme control and shows the transfer of uncleaved substrate. Lanes 2-5 The Cleavease^TMReactions containing 10, 50, 100 or 250 ng of BN enzyme respectively Contains reaction products from the liquid.   These results indicate that even if the amount of enzyme used in the reaction Regardless, the same cleavage pattern was obtained using a 157 nucleotide tyrosinase DNA substrate. Is obtained. Therefore, this method is mostly used in research laboratories. Ideally suited for clinical labs with uncontrolled reaction conditions . F) Consistent cleavage patterns are obtained with different DNA preparations   To demonstrate that the same cleavage pattern is consistently obtained from a particular substrate, Of a 157 base fragment (SEQ ID NO: 34) derived from the sense strand of exon 4 of the synase gene Several different preparations were made. The substrate was prepared as described in Example 8b. So Three separate PCR reactions were performed on different days. One of these PCR samples One was split into two, one aliquot was gel purified on the day it was generated, and the other aliquot was The plate was stored at 4 ° C. overnight and gel purified the next day.   The cleavage reaction was performed as described in Example 8b. Acrylamide sample And run as described in Example 8b. The obtained autoradiograph FIG.   In FIG. 36, the lane marked “M” contains a biotinylated molecular weight marker. Have. Set 1 contains preparation no. 1 in the absence (-) or presence of the enzyme Contains the product from the cleavage reaction performed in (+). Set 2 contains formulation no. 2 From the cleavage reaction carried out in the absence (-) or presence (+) of the enzyme using Contains a product. Set 3 contains formulation no. 3 in the absence of the enzyme (-) Contains the product from the cleavage reaction performed in the presence (+). Preparation no. 3 is key Product no. 2 from Preparation no. 3 is the preparation no. Gel purification after 1 day Other than that, it is the same. Set 4 contains formulation no. 4 in the absence of enzyme Contains products from cleavage reactions performed in (-) or in the presence (+). these The same pattern of cleavage products was generated from separately prepared substrate samples I have.   These results indicate that separately prepared preparations of the 157 nucleotide DNA fragment were identical. This indicates that a single cleavage pattern was obtained. Therefore, Cleavase^TM The reaction is not affected by small differences that exist between the substrate preparations.                                 Example 12         Using the sense or antisense strand of the tyrosinase gene                           Point mutation detected   Using the sense (coding) or antisense (non-coding) strand of the gene fragment Cleavage to create a unique pattern (ie, fingerprint) of cleavage products Boase^TMThe ability of the enzyme was examined.   Sense nucleotide of 157 nucleotide fragment derived from wild-type tyrosinase gene (SEQ ID NO: No. 34) or the single-stranded DNA substrate corresponding to the antisense strand (SEQ ID NO: 35). , Prepared using non-control PCR as described in Example 8a. Sense strand wild type The substrate contains a biotin label at the 5 'end. The antisense strand has a fluorescein Contains an in-label.   Senses a 157 nucleotide fragment from the 419 mutant tyrosinase gene The corresponding single-stranded DNA substrate (SEQ ID NO: 41) was prepared in Example 9 using non-control PCR. Prepared as described. Sense mutant 419 mutant substrate is biotin labeled at the 5 'end It contains.   Antisense of a 157-nucleotide fragment from the 419 mutant tyrosinase gene. A single-stranded DNA substrate corresponding to the strand (SEQ ID NO: 52) was run using uncontrolled PCR Prepared as described in Example 9. However, 100 pmol of fluorescein-labeled anti Sense primer (SEQ ID NO: 30) and 1 pmol of biotin-labeled sense primer -(SEQ ID NO: 29) was used. The resulting antisense strand 419 mutant substrate is 5 ' Contains a fluorescein label at the end.   Senses a 157 nucleotide fragment from the 422 mutant tyrosinase gene The corresponding single-stranded DNA substrate (SEQ ID NO: 42) was prepared in Example 9 using non-control PCR. Prepared as described. Sense mutant 422 mutant substrate is biotin labeled at the 5 'end It contains.   Antisense of a 157 nucleotide fragment from the 422 mutant tyrosinase gene A single-stranded DNA substrate corresponding to the strand (SEQ ID NO: 53) was run using uncontrolled PCR Prepared as described in Example 9. However, 100 pmol of fluorescein-labeled anti Sense primer (SEQ ID NO: 30) and 1 pmol of biotin-labeled sense primer -(SEQ ID NO: 29) was used. The resulting antisense strand 422 mutant substrate is 5 ' Contains a fluorescein label at the end.   After asymmetric PCR, the single-stranded PCR product was gel purified as described in Example 8. Prepared, precipitated and resuspended in 40 μl of TE buffer.   Perform the cleavage reaction as described in Example 9 and generate the product as described in Example 8a. The product was fractionated by electrophoresis. After electrophoresis, transfer the DNA to a nylon membrane, Conjugate and anti-fluorescein antibody (Fab) -alkaline phosphatase conjugate And visualized using CDPStar as described in Example 8a. Obtained Oh The radiograph is shown in FIG.   In FIG. 37, the lanes marked “M” are as described in Example 8. Contains prepared biotinylated molecular weight markers. Lanes 1-6 are wild type 419 And 422 mutants contain biotinylated sense strand substrates from 157 nucleotide fragments I do. Lanes 1-3 show enzyme-free pairs for wild-type, 419 and 422 mutant fragments. Respectively. Lanes 4-6 show wild type, 419 and 422 mutant fragments Cleaving the sense strand^TMReaction product obtained by incubation with BN enzyme Stuffs. Lanes 7-12 show wild type, 419 and 422 mutant 15 Contains a fluoresceinated antisense strand substrate from a 7 nucleotide fragment. Les 1 to 3 are "enzyme-free" for wild-type, 419 and 422 mutant fragments, respectively. "Contains control. Lanes 4-6 show the results for wild type, 419 and 422 mutant fragments. Cleaving the antisense strand^TMReaction products obtained by incubation with BN enzyme Each composition is contained.   As expected, when using sense or antisense fragments, wild-type, 41 Separate but unique patterns of cleavage products for the 9 and 422 mutant fragments Generated. Uses either the sense or antisense strand of the gene as substrate The ability to do is advantageous. Because, under certain reaction conditions, two One of the strands produces a more desirable band pattern (ie, the cleavage product Do not collect at one end of the gel, but spread over the entire length of the gel). This is because that. Alternatively, one strand is more convenient to create significant structural changes. This is because they may have well-located mutations. This means that More important in the analysis of long DNA substrates with mutations near their ends. Could be. In addition, detection using both strands can serve as confirmation of sequence changes. One.                                 Example 13Detection of mutations in the human β- globin gene using enzymatic cleaving ase ^TM   The results shown in Examples 8 to 12 are^TM157 nucleotides using the reaction For a single base change in the tyrosinase gene fragment ranging from Indicates that it can be issued. Cleavease^TMReaction is mutation detection The second model system was examined to show that it can be widely applied to.   The human β-globin gene has a number of differences, including sickle cell anemia and β-thalassemia. It is known that mutations occur in hemoglobinopathies. these Diseases generally have a small number (1-4) in the DNA sequence of the wild-type β-globin gene. With nucleotide changes [Orkin, S.H. and Kazazian, H.H., Jr. (1984) Annu . Rev. Genet. 18: 131 and Collins, F.S. and Weissman, S.M. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31: 315]. , Less in the β-globin gene And 47 different mutations have been identified which cause β-thalassemia You.   Cleavease^TMReactions were used to convert the three β-globin mutants to wild-type β-g Robin gene [Lawn, R.M. et al., (1980) Cell 21: 647]. Mutant 1 Contains a nonsense mutation at codon 39. The wild type sequence at codon 39 is CAG It is. The sequence of mutant 1 at this codon is TAG [Orkin, S.H. And Goff, S.C. (1981) J.A. Biol. Chem. 256: 9782]. Mutant 2 is the primary transcript Has a T to A substitution at codon 24 resulting in improper splicing of [Gol dsmith, M.E. et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2318]. Mutant 3 Introduces a deletion of two A residues that results in a reading frame mobility difference at codon 8 To have. Mutant 3 also has a silent C to T substitution at codon 9. [Orkin, S.H. and Goff, S.C. (1981) J.A. Biol. Chem. 256: 9782]. A) Preparation of wild-type and mutant globin gene substrates   From the wild-type and mutant β-globin genes described above, a single-stranded substrate DNA was prepared. It was prepared as follows. Place bacteria with the appropriate plasmid on the antibiotic plate. Treaked and grown overnight at 37 ° C. (Wild type plasmid and mutant 3 Bacteria with the containing plasmid were grown on tetracycline plates. Bacteria harboring plasmids containing mutant 1 and mutant 2 sequences Grow on picillin plates. Next, colonies from these plates were And the Wizard Minipreps DNA Purification System (Promega Corp., Ma. dison, WI). Colonies derived from the above kit In 200 μl of “cell resuspension buffer”. According to the manufacturer's protocol To extract DNA. A final yield of approximately 2.5 μg was obtained for each plasmid.   In 50 μl of 1 × PCR buffer, 5 ng of plasmid DNA, 25 pmol each of 5 ′ end bio KM29 primer (SEQ ID NO: 54) and 5'-terminal fluorescein-labeled RS42 Immer (SEQ ID NO: 55), 50 μM of each dNTP and 1.25 units of Taq DNA polymer The polymerase chain reaction containing the 536 (wild type, mutant 1 and 2) or 534 (mutant 3) nuclei, respectively The reotide fragment was amplified. The reaction solution is overlaid with two drops of light mineral oil, Heat at 95 ° C for 30 seconds using a cycler (MJ Research, Watertown, MA) and 30 seconds Cooled to 55 ° C and heated to 72 ° C for 60 seconds, this was repeated 35 times. Wizard PCR Clean Using the up kit (Promega Corp., Madison, WI), remove the products of these reactions into residual dNTPs. And purified from the primers, and the double-stranded DNA was dissolved in 50 μl of TE.   To make a single-stranded copy of these DNAs, 1 μl of double-stranded PCR DNA Was used as a template and the above PCR was repeated, omitting the RS42 primer. this The products of the asymmetric PCR are combined with the first P to identify the location of the double stranded DNA product. 6% polyacrylamide gel in 0.5X TBE buffer (29: 1 cross-linked) with CR DNA ) Directly loaded. After electrophoretic separation, stain with ethidium bromide. DNA was visualized by color. And the changed transfer as compared to the double-stranded DNA. The single-stranded DNA identified by the mobility was cut out and gel-slurried by passive diffusion overnight. 0.5M NH from chair_FourIt eluted in a solution containing OAc, 0.1% SDS and 0.2 mM EDTA. Eta The product was recovered by knol precipitation and dissolved in 40 μl of TE.   SEQ ID NO: 56 shows the sequence of a 536 nucleotide fragment derived from wild-type β-globin gene. You. The sequence of the 534 nucleotide fragment from mutant 3 is shown in SEQ ID NO: 57. suddenly SEQ ID NO: 58 shows the sequence of the 536 nucleotide fragment derived from Mutant 1. Mutant 2 The sequence of the derived 536 nucleotide fragment is shown in SEQ ID NO: 59. B) Optimization of cleavage reaction using wild-type β-globin substrate     An array of cleavage products with very different mobilities from β-globin substrates The optimal conditions (salt concentration, temperature) for formation were confirmed. The most uniform strength between bands Conditions that create the most widely dispersed array of cleavage patterns The formation of such a band is due to the difference between the wild-type and mutant substrates. Help detection). This experiment was performed in the absence of KCl or in the presence of 50 mM KCl. The cleavage reaction was performed at different temperatures using a wild-type β-globin substrate (SEQ ID NO: 56). Applying.   For each KCl concentration to be tested, DNA, CFLP^TM3 containing buffer and salts 0 μl of the master mixture was prepared. For a “0 mM KCl” reaction, the mixture is 1 .7mM (1mM in final reaction) MnCl_TwoAbout 50 fm in 30 μl of 10 mM MOPS, pH 8.2 containing ol containing the single-stranded 5 'biotinylated 536mer PCR DNA from plasmid pHBG1 . The “50 mM KCl” mixture contained 83.3 mM KCl in addition to the above components. these Dispense 6 μl of the mixture into labeled reaction tubes and keep on ice until use. Existed. Enzyme dilution cocktail is MnCl_Two450 ng of 10 mM MOPS, pH 8.2 Leavease^TMBN.   Cleavage reactions were performed at 60, 65, 70 and 75 ° C. For each temperature to be tested, KC The pair of tubes, with and without l, are heated to 95 ° C for 5 seconds and then selected. Cooled to the specified temperature. The reaction was then immediately started by adding 4 μl of enzyme cocktail. Started. An additional pair of tubes is used for the 75 ° C test, and these tubes are Is MnCl instead of adding enzyme_Two4 μl of 10 mM MOPS, pH 8.2 containing no. Oh No ill overlay was used. All reactions were allowed to proceed for 5 minutes, then 8 μl of stop buffer Stopped by addition of liquid. Completed and starting reactions , Stored on ice until all reactions were performed. Heat the sample to 72 ° C for 2 minutes, 5 μl of the reaction solution was dissolved in a buffer containing 0.5X TBE and 6% polyacrylic acid containing 7M urea. Fractionation was performed by electrophoresis in a luamide gel (19: 1 cross-linking).   After electrophoresis, remove the gel plate and leave the gel flat on one plate I did it. Positively charged with 0.2 mm pores pre-wetted with water Of exposed gel exposed nylon film (NYTRAN, Schleicher and Schuell, Keene, NH) I put it on the top. All air bubbles were removed. Next, apply 2 pieces of 3MM filter paper (Whatman) Replace the other glass plate, and place the sandwich It is sandwiched between inder clips. The migration proceeded overnight. Carefully remove the membrane after transfer. And air dried. After completely drying, 1.2X Sequenase Images 1 cm membrane in blocking buffer (United States Biochemical)^TwoPer buffer above The solution was washed with 0.3 ml. Washing was performed for 30 minutes. Streptavidin-Alka Rephosphatase conjugate (SAAP, United States Biochemical) diluted 1: 4000 The solution was directly added to the above-mentioned blocking solution until the mixture became, and stirred for 15 minutes. Short time membrane with water Rinse, then 1 cm membrane^Two1X containing 0.5 ml of 0.1% sodium dodecyl sulfate (SDS) Using SAAP buffer (100 mM Tris-HCl, pH10; 50 mM NaCl) for 3 times per 5 minutes Washed twice. The membrane was briefly rinsed with water between each wash. Next, 1X SA without SDS AP / l mM MgCl_TwoWash the membrane once with In a bag that can be Using a sterile pipette, for alkaline phosphatase CSPD^TMOr CDP-Star^TM(Tropix, Bedford, MA) Add 5 ml of chemiluminescent substrate to this bag. And allowed to spread over the entire membrane for 2-3 minutes. CSPD^TMMembrane treated with XRP X-ray film Incubated for 30 minutes at 37 ° C. prior to the first exposure to (Kodak) (60 minutes). CDP-Star^TMDoes not require pre-incubation and the first exposure is 10 minutes. Adjusted exposure time as needed for resolution and clarity . The results are shown in FIG.   In FIG. 38, the lane marked “M” contains the molecular weight marker . Lanes 1-5 contain reaction products from reactions performed in the absence of KCl. . Lane 1 contains the reaction product of a reaction performed at 75 ° C. without enzyme. Lane 2 ~ 5 contain the reaction products of the reactions performed at 60, 65, 70 and 75 ° C respectively. Lanes 6-10 contain the reaction products of the reaction performed in the presence of 50 mM KCl. Les Lane 6 contains the reaction product of the reaction performed at 75 ° C. without the enzyme. Lanes 7-10 are It contains the reaction products of the reactions performed at 60, 65, 70 and 75 ° C. respectively.   In general, the preferred pattern of cleavage products is when the reaction contains 50 mM KCl. Was generated if At any of the temperatures tested, as seen in lanes 7-10, K Compared to the reaction performed in the absence of Cl (lanes 2-5: more cleavage products Swarming on top of the gel near the unbroken substrate), reaction formation The material was more widely dispersed in reactions containing 50 mM KCl. Lane 7 in FIG. As can be seen in Figure 5, the cleavage reaction performed at 65 ° C with 50 mM KCl Compared with the pattern in the cleavage product, where the product is Produced a pattern.   The results obtained in this experiment indicate that 536 nucleotides from the wild-type β-globin gene Optimal cleavage conditions for the dosense strand fragment (SEQ ID NO: 56) are 1 mM MnCl at 60 ° C._TwoAnd 50 It was confirmed to be 10 mM MOPS, pH 8.2, containing mM KCl. C) Optimization of the cleavage reaction using two mutant β-globin substrates   From the results obtained in a) and b) above, the openness when β-globin substrate is used 60 ° C was selected as the optimal temperature for the cleavage reaction. When using a wild-type substrate, 50 mM KCl Performing the cleavage reaction in the presence produces an optimal pattern of cleavage products. Wild type Patterns generated when using both mutated and mutant β-globin substrates The effect of KCl on the effect of KCl on the wild-type and mutant β-globin substrates was then examined. The optimal salt concentration that allowed the comparison of was determined.   Mutant 1 (SEQ ID NO: 58) and mutant having a length of 536 nucleotides The single-stranded substrate corresponding to the 2 (SEQ ID NO: 59) mutation is as described in a) above. Prepared. These two mutants have the wild-type sequence and one nucleotide each. Only different. These two differ from each other by two nucleotides.   For each substrate tested, DNA, CFLP^TMBuffer and MnCl_Two39 μl containing A master mixture was prepared. These mixtures contain approximately 500 fmol of single-stranded 5 'biotin. 536 nucleotide substrate DNA, 1.54 mM MnCl_Two(Final concentration 1mM MnCl_TwoGive) It contained 39 μl of 10 mM MOPS, pH 8.2. Label these mixtures 6.5 μl aliquots were dispensed into the reaction tubes. Next, add 10 mM each to each aliquot. 0.5 μl of 200 mM KCl was added for a final KCl concentration (eg, 40 2.0 μl was added to the mM reaction tube). And dH_Two9 μl all volumes using O I made it. No oil overlay was used. Heat the reaction to 95 ° C for 5 seconds, then 65 ° C And cooled. 1 μl of dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% NP40 , 0.5% Tween 20, 10 mg / μl BSA)^TMAdd BN This immediately started the cleavage reaction. After all reactions have proceeded for 5 minutes Stopped by adding 8 μl of stop buffer. Heat the sample to 72 ° C for 2 minutes Then, 5 μl of each reaction solution was dissolved in a buffer containing 0.5 × TBE, and 6% Fractionation was performed by electrophoresis in a acrylamide gel (19: 1 cross-linking).   After electrophoresis, remove the gel plate and place one gel on the gel as described above. Lon film was overlaid. The DNA is transferred to a membrane and the membrane is treated as described in a) above. And exposed to X-ray film. FIG. 39 shows the obtained autoradiograph. .   In FIG. 39, the lane marked “M” contains the molecular weight marker . Lanes 1, 3, 5, 7, 9 and 11 are 0, 10, 20, 30, 40 or 50 m respectively Contains reaction products from cleavage reaction using mutant 1 substrate in the presence of M KCl You. Lanes 2, 4, 6, 8, 10 and 12 are 0, 10, 20, 30, 40 or 5 respectively Contains reaction products from cleavage reaction using mutant 2 substrate in the presence of 0 mM KCl I do.   FIG. 39 shows the cleavage products generated from each mutant as KCl concentration increased. Pattern varies, but a distinct pattern is generated from each mutant and tested. At any KCl concentration, the difference in band pattern between the two mutants Indicates that it can be seen. In the intermediate salt concentration range (10-20 mM KCl), large cleavage The band disappears and a small molecular weight band appears (lanes 3-6). High salt concentration Comminant loss of small molecular weight bands (30-50 mM KCl) A large molecular weight cleavage band reappears with (lanes 7-12). Shown in Figure 39 From the results, reaction conditions including the use of 50 mM KCl were selected as optimal reaction conditions. about In the intensity of the band appearing at the position of 200 nucleotides (see the arrow in FIG. 39) A clear difference can be seen between the two mutant substrates under these reaction conditions. It is. D) Cleavase enzyme^TMIs unique from wild-type and mutant β-globin substrates Generates cleavage products.   From the experiments performed above, it was found that wild-type or mutant β-globin substrates In this case, it was confirmed that the optimal reaction conditions were using 50 mM KCl and a temperature of 60 ° C. Therefore, the wild-type substrate (SEQ ID NO: 56) was changed to mutant 1 (SEQ ID NO: 58) For comparison with the variant 2 (SEQ ID NO: 59) and mutant 3 (SEQ ID NO: 57) substrates These conditions are used to allow comparison of the cleavage patterns generated Was done.   Wild type, mutant 1, mutant 2 and mutant 3β-globin genes For 534 or 536 nucleotides of single-stranded substrate DNA as described in a) above. Was prepared. The cleavage reaction was performed as follows. 1.1mM MnCl_Two(Final density 9 μl of 1.10 mM MOPS, pH 8.2 containing 5 mM mM KCl (final concentration 50 mM). Reaction tubes containing about 10 fmol of each DNA substrate in (final concentration 1X) were prepared. . A "no enzyme" or uncleaved control was made for each substrate DNA. The uncut control is In order to balance the signal seen on the autoradiograph, It contained 1/3 of the DNA (about 33 fmol) of the reaction solution.   The tube is heated to 95 ° C. for 5 seconds, cooled to 65 ° C., and 1 μl of Cleavase^TM The addition of BN enzyme (50 ng / μl in 1 × dilution buffer) directly cleaves the cleavage reaction. Started immediately. Uncut controls received 1 μl of 1 × dilution buffer.   The reaction was allowed to proceed for 5 minutes and then stopped by the addition of 8 μl of stop buffer. Sun Heat the pull to 72 ° C for 2 minutes and dissolve 5 μl of each reaction in buffer containing 0.5X TBE. Fractionated by electrophoresis in disassembled 6% polyacrylamide gel containing 7M urea (19: 1 cross-linking) did.   After electrophoresis, the gel plate was removed and one gel was added to the gel as described above. Lon film was overlaid. Transfer the DNA to a membrane and treat the membrane as described in b) above. And exposed to X-ray film. FIG. 40 shows the obtained autoradiograph. .   FIG. 40 shows two panels. The first panel is from the above cleavage reaction Shows the reaction product of Uncleaved controls are shown in lanes 1-4, cleavage products in lanes 5-6. The second panel is seen between the substrate DNA at the top of the gel Lanes 5-6 are enlarged to better show different band patterns.   In FIG. 40, the lanes marked “M” are as described in Example 8. Contains prepared biotinylated molecular weight markers. Lanes 1 to 4 are wild type, Uncleaved mutant 1, mutant 2 and mutant 3 β-globin substrate Each contains a control. Lanes 5-8 are wild type, mutant 1, mutant 2 and mutant 3β-globin substrates, respectively, containing cleavage products.   From the results shown in FIG.^TMBN enzyme was tested for each β-globin Generates a unique cleavage product pattern from the substrate. Wild type difference in band pattern And found between each mutant. Different band patterns for each mutant And not only the discrimination between wild-type and mutant, but also It is also possible to distinguish them from mutants.   The results shown here for the β-globin gene and for the tyrosinase gene The results shown above are for the detection of mutated genes in the CleavaseTM reaction. It offers a powerful new tool.                                 Example 14             RNA substrate processing generates unique cleavage patterns   The present invention provides a method for generating a unique band pattern characteristic of a particular substrate. It is intended for 5 'nuclease cleavage of single-stranded or double-stranded DNA substrates. Cleavure -Se^TMAbility of 5 'nuclease activity of BN enzyme to utilize RNA as substrate nucleic acid Is shown below. This experiment was performed using appropriate conditions (dropping the pH from 8.2 to 6.5, Reduces the degradation of RNA substrates mediated by cancer), generates cleavage patterns Has shown that RNA can be used as a substrate for Perform the experiment as follows did.   RNA transcripts internally labeled with biotin were made and used as substrates. Step Rasmid pGEM3Zf (Promega) was digested with EcoRI. EcoRI cuts the plasmid and Generate a linear template. Riboprobe Gemini System kit from Promega Corp. Using SP6 transcription of the linearized template described above, RNA of 64 nucleotides in length A transcript (SEQ ID NO: 60) was made. Followed the manufacturer's instructions but in the reaction Internally labeled by replacing 25% of UTP with biotin-UTP (Boehringer Mannheim) The resulting transcript was produced. After the transcription reaction (1 hour, 35 ° C), RQ1 RNase-free DNA DNA template by treatment with se (from Riboprobe kit, used according to manufacturer's instructions) Was removed. Then, the RNA is recovered and 2M NH_FourIn the presence of OAc and ethanol The sample was purified by precipitation twice. 70% of the obtained RNA pellet Rinse with ethanol, air dry, 40 μl of 10 mM Tris-HCl, pH 8.0 and 1 mM E Resuspended in DTA.   Cleavage reaction solution is 10 μl of 1X RNA-CFLP^TMBuffer (10 mM MOPS, pH 6.3) and 1 mM M gCl_TwoOr MnCl_Two1 μl of the above RNA substrate and 50 ng of the enzyme cleavase^TM BN. Put all components except the enzyme in the reaction solution, and 45 ° C for 30 seconds Heated. 1 μl of dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% NP40, 50 ng of enzyme cleavease in 0.5% Tween 20, 10 μg / ml BSA)^TMAdd BN To start the reaction. After allowing the reaction to proceed for 10 minutes, 8 μl of stop buffer Stopped by addition of liquid. Heat the sample to 72 ° C for 2 minutes and transfer 5 μl of each reaction. A 10% polyacrylamide gel containing 7M urea dissolved in a buffer containing 0.5X TBE ( 19: 1 cross-linking).   After electrophoresis, the gel plate was removed and the gel was applied to the gel as described in Example 8b. Nylon membranes were overlaid. Transferring the DNA to a membrane as described in Example 8b, Dry the membrane, wash with 1.2X Sequenase Images blocking buffer and 1X SAAP buffer Treated with Lumiphos-530 (United States Biochemical) or Quantum Y Reaction with an ield chemiluminescent substrate (Promega) and exposure to X-ray film. Obtained Oh The radiograph is shown in FIG.   In FIG. 41, the lane marked "M" contains the molecular weight marker. Les Lane 1 contains the no enzyme control and shows the transfer of uncleaved substrate. Lane 2 and And 3 are Cleavase^TMMgCl in the presence or absence of BN enzyme_TwoContains Generation from RNA substrate incubations performed in different buffers Material. Lanes 4 and 5 are Cleavase^TMIn the presence of BN enzyme or In the absence of MnCl_TwoRNA substrate inks each performed in a buffer containing It contains the reaction product from the incubation. MnCl_TwoWith RNA substrate in the presence of When the enzyme was incubated (lane 5), a pattern of cleavage products was seen. You.   These results indicate that for sequence changes (eg, point mutations, deletions, substitutions) Cleavase to probe NA substrates^TMBN enzyme can be used It is shown that. This ability is very large (eg, 10 Allows testing of genes with introns (greater than kb). Spliced RNA transcripts represent a simpler target for mutation scanning. Further, R The structural (ie, folding) information obtained by the cleavage of NA is Hybridization to unstructured regions or ribozyme probing May also be useful in targeting targets. Furthermore, the cleavage reaction happens very quickly So, Cleavease^TMVarious types of RNA folding using the BN enzyme, and It is possible to study the kinetics at which folding occurs.                                 Example 15         In both of Cleve-ase ^TM BN enzyme and Taq polymerase Five' Nuclease activity also generates unique cleavage patterns using double-stranded DNA substrates   Cleavase generating cleavage pattern using single-stranded DNA template^TMBN enzyme And the ability of both Taq polymerase were examined. The substrates used in this experiment are Wild type (SEQ ID NO: 43) or 422 mutant (SEQ ID NO: 44) tyrosinase It was a 378 nucleotide fragment derived from the gene. These single-stranded substrates are described in Example 10a. Generated as described.   The cleavage reaction was performed as described in Example 10b. However, note half of the reaction solution. Cleavease as stated^TMReactions cut with BN enzyme and set in parallel The solution was cleaved with Taq polymerase. Taq polymerase reaction is 10 mM MOPS, pH8.2 Consisted of 1.25 units of Taq polymerase. As described in Example 10b, The reaction products were separated by electrophoresis, and an autoradiograph was prepared. This auto The radiograph is shown in FIG.   In FIG. 42, the lane marked “M” contains a biotinylated molecular weight marker. Have. Lanes 1 and 2 are cleaved^TMFields cleaved by BN enzyme Contains the native or 422 mutant substrate, respectively. Lanes 3 and 4 are Taq ports Contains wild-type or 422 mutant substrates, respectively, cleaved by limerase I do.   Fig. 42 shows the cleaving^TMBoth BN enzyme and Taq polymerase Generates a characteristic set of cleavage bands for quality, wild type and 422 mutant This indicates that the substrate can be distinguished. These two enzymes are associated with each template. To produce a similar but different array of bands.   These results are^TM5 for both BN enzyme and Taq polymerase '' Shows that nucleases are useful for performing the cleavage reaction of the present invention. You. Cleavage by Taq polymerase means that the substrate is generated by PCR and Any intervening refinement other than removal of excess nucleotides using rukariphosphatase If no manufacturing process is employed, one will find use.                                   Example 16                                 Multiple cleavage reactions   The examples described so far use single-stranded DNA and RNA substrates using a cleavage reaction. Shows that a distinctive set of cleavage products can be produced from C. The ability of the cleavage reaction to use a double-stranded DNA template was examined. For many applications, two Performing the cleavage reaction directly with the strand PCR product requires a single-stranded No need to isolate quality and would be ideal. In addition, many reaction tubes It would be advantageous to be able to analyze the substrate ("multiplex" reactions).   5 'biotin label on sense strand and fluorescein label on antisense strand The cleavage reaction was performed using the supported double-stranded template. Prior to cleavage, double-stranded substrate Was denatured. The double-stranded substrate was cleaved using Taq polymerase. T for this experiment aq polymerase was used. Because this is Cleavease^TMThan the BN enzyme It has a weak duplex-dependent 5 '→ 3' exonuclease activity, and Rase is Cleavase^TMFrom DNA double strand regenerated as efficiently as BN enzyme This is because the 5 'end label is not removed. Therefore, the signal lost in the reaction Less is.   The substrate used was the wild-type tyrosinase gene (SEQ ID NO: 34), a 419 mutant ( It was a 157 bp fragment from the SEQ ID NO: 41) or 422 mutant (SEQ ID NO: 42). wild The type fragment is as described in Example 8a and the 419 mutant fragment is as described in Example 8a. And the 422 mutant fragment was generated using PCR as described in Example 9. Was done. The sense strand primer (SEQ ID NO: 29) contains a 5 'biotin label, The chisense primer (SEQ ID NO: 30) contains a 5 'fluorescein label and each strand has This results in the production of a double-stranded PCR product that is labeled with a different label. PCR generation The material was gel purified as described in Example 8a.   The cleavage reaction was performed as follows. In a reaction tube, 5 μl of 10 mM MOPS, pH 8.2 , 1mM MnCl_TwoAbout 100 fmol of the double-stranded DNA substrate in was loaded. Add one drop of this solution Layered with mineral oil. Heat the test tube to 95 ° C for 30 seconds and add 1 unit of Taq Polymerase (Promega) was added. The uncleaved control was 5 μl of 10 mM MOPS, pH 8.2, 1 mM MnCl_TwoIt contained 33 fmol of double-stranded DNA substrate. Cool the reaction to 65 ° C And incubated at this temperature for 15 minutes. Reaction by adding 8 μl of stop buffer Was stopped. Heat the sample to 72 ° C for 2 minutes, add 5 μl of each reaction and 0.5X TBE In 10% polyacrylamide gel containing 7M urea dissolved in buffer containing (19: 1 cross-linking) Was fractionated by electrophoresis. You can have two nylon membranes with the whole set of reactions Thus, the entire set of reactions was loaded onto the gel using two sets of samples. Nylon After transfer to the membrane (performed as described in Example 8a), the membrane was cut in half. one of One half was probed with a streptavidin-alkaline phosphatase conjugate. The biotinylated sense strand product was visualized (as described in Example 8a). Film residue Half of the protein using anti-fluorescein antibody-alkaline phosphatase conjugate To visualize the fluorescein-labeled antisense strand product (described in Example 5). As described). For biotin-labeled and fluorescein-labeled DNA, Blots were visualized using the chemiluminescent procedure described in Examples 8a and 5, respectively. So 43 are shown side by side in FIG.   In FIG. 43, the lanes marked “M” are as described in Example 8. Contains prepared biotinylated molecular weight markers. Lane marked "M2" Digests pUC19 with MspI followed by Klenow treatment for blunt end Contains a molecular weight marker produced by Next, the terminal transferer Fluoresceinated dideoxynucleotides (Bo ehringer Mannheim). Lanes M-1 and 1-6 contain biotinylated DNA. Developed using the protocol described above. Lanes 7-12 and M2 are fluorescein standards Developed using protocol for DNA recognition. All lanes have both substrates Note that there are chains. In certain development protocols, only one strand is Be visualized.   In FIG. 43, lanes 1-3 and 7-9 are wild type, 419 or 422 abruptly altered. Contains "no enzyme" or uncleaved controls, each with a heterologous substrate. Lane 4 ~ 6 and 10-12 are open-ended from wild-type, 419 or 422 mutant substrates, respectively. Contains crack products. Cleavage products for each strand of each of the three substrates tested A unique pattern can be seen. Thus, one reaction is equivalent to the three tyros tested. Unique fingers from each sense or antisense strand of the synase fragment -It made it possible to generate prints.   The results shown in FIG. 43 were obtained by denaturing the DNA fragment before performing the cleavage reaction. This indicates that a cleavage pattern can be generated from a double-stranded DNA fragment. FIG. In 3, the cleavage pattern is one round of heating for denaturation and cooling for cleavage. Note that most of the substrate remains in uncleaved form. I want to be. This reaction involves multiple cycles of denaturation and cleavage using a thermocycler. Would be acceptable. Such repetitive conditions are known to Will increase the signal intensity seen for the object. Standard PCR buffer (50m M KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl_Two, 0.01% gelatin) The substrate generated by PCR is treated to remove residual dNTPs (eg, alkaline Addition of phosphatase) and Mn²⁺Can be supplied. Under these conditions The cleavage reaction is performed using both strands of one or more products generated in the PCR. Can be implemented. Such a protocol reduces sample preparation to a minimum. , Resulting in both time and cost savings.   The above example also demonstrates that two different substrates can be analyzed in one reaction. It shows that this allows "multiplexing" of the cleavage reaction.                                 Example 17         Optimization of manganese ion concentration for cleavage of double-stranded DNA substrates   As described above, restriction fragments or segments generated by equilibrium or symmetric PCR It may be desirable to carry out the cleavage reaction using double-stranded DNA substrates such as Not. Mn in cleavage reaction using double-stranded DNA substrate^2-The effect of changing the concentration of Examined. The results shown below show that when a double-stranded DNA substrate is used for the cleavage reaction, Mn compared to single-stranded DNA substrate²⁺Shows that the optimum concentration becomes lower.   From tyrosinase mutants 419 (SEQ ID NO: 27) and 422 (SEQ ID NO: 71) Two 157 bp long double-stranded (ds) DNA substrates were used to excise the human tyrosinase gene. Prepared as described in Example 16 by PCR amplification of the Son 4 region. 419 and 4 The sense strand of the 22 tyrosinase mutant substrate has a biotin label at the 5 'end after PCR. I knew. The PCR product was gel purified as described in Example 8a.   The cleavage reaction was performed as follows. About 40 fmoles of dsDNA groups in 10 μl of water The quality was placed in a reaction tube. PTC-100^TMProgrammable Thermal Controller (MJ Rese arch, Inc.) to denature the DNA by placing the test tube at 95 ° C. for 10 seconds. 1 μl Leavease^TMBN enzyme (25 ng in 1 × dilution buffer) and reaction mixture (final volume 20 μl) MnCl in l)_TwoFinal concentration of 0.5mM, 0.25mM, 0.15mM, 0.1mM, 0.05Mm or 0m Different concentrations of MnCl to be M_Two10 μl of 2X CFLP containing^TMBuffer (pH 8.2) The reaction was started by the addition. After mixing, cool the sample immediately to 65 ° C. And incubated at this temperature for 5 minutes. Place sample on ice and 10 μl The reaction was stopped by adding a stop buffer. Keep sample at 85 ° C for 30 seconds Heat and add 10 μl of each reaction to a buffer containing 0.5 M TBE containing 7 M urea. Fractionation was performed by electrophoresis in a 0% polyacrylamide gel (19: 1 cross-linking).   After electrophoresis, remove the gel plate as described in Example 8a, and remove the nylon membrane. Covered. The DNA was transferred to a membrane as described in Example 8a, the membrane was dried and 1X Seq Wash with uenase Images blocking buffer (USB), treat with 1X SAAP buffer, and CDP-Star^TM(Tropix) and exposed to X-ray film. Auto obtained The radiograph is shown in FIG.   In FIG. 44, the lane labeled "M" was prepared as described in Example 8. Including molecular weight markers. Lanes 1-6 show the products resulting from cleavage of the 419 mutant. And lanes 7-12 contain the product resulting from cleavage of the 422 mutant. 0. 5 mM (lanes 1, 7): 0.25 mM (lanes 2, 8): 0.15 mM (lanes 3, 9): 0.1 mM (lanes 4, 10) : 0.05 mM (lanes 5, 11) and 0 mM MnCl_Two(Lanes 6, 12) in 10 mM MOPS, pH 8.2 In the following, a reaction product generated by cleavage of a dsDNA substrate is shown.   The results shown in FIG. 44 are similar to those for cleavage of the ssDNA substrate (Example 11 (a ) And FIG. 31), indicating that no cleavage was observed without the divalent cation. . Optimal cleavage of the dsDNA substrate (ie, the most pronounced cleavage fragment pattern) Is 0.25mM MnCl_TwoWas seen in the presence of This optimal concentration is Much lower than the optimal concentration obtained with Example 11 and FIG. The cleavage of the A substrate is 1 mM MnCl_TwoIndicates that it is optimal). FIG. 44 shows a dsDNA group. Quality cleavage efficiency is MnCl_TwoThis effect decreases with decreasing concentration, and this effect is_Two This may be because the efficiency of the enzyme decreases as the concentration decreases.   FIG. 44 shows a high concentration of MnCl_TwoUsing (0.5 mM, lanes 1 and 7) for the cleavage reaction, It shows that the cleavage pattern of the dsDNA substrate clearly disappears. The result is a cleavage In contrast to the results obtained when the reaction was performed on single-stranded DNA (ssDNA) substrates is there. Example 11 (a) shows that efficient cleavage of the ssDNA substrate is 1 mM MnCl_TwoGet in And Mn²⁺Almost no change in cleavage pattern is observed between 1 and 4 mM Indicates that   High concentration of MnCl_TwoSignal when cleaving a dsDNA substrate in a buffer containing The disappearance can be explained as follows. The presence of a high concentration of divalent ions causes the cleavage reaction Promotes recombination of the DNA strand of the ds substrate during the course of the reaction. Enzyme Cleavase (Clea vase)^TMBN can cleave a dsDNA substrate from the 5 'end (ie, This enzyme removes short exonuclease DNA fragments from the 5 'end. : See Example 5). As a result of this cleavage, the label is apparently removed from the substrate DNA (Since the DNA contains a 5 'end label). Very short D with 5 'end label NA fragments can run out of the gel or are not efficiently transferred to the membrane. Cannot be visualized.                                 Example 18                   Automated cleavage pattern detection is possible   Using a fluorescently labeled substrate in combination with a fully automated DNA sequencer Detects characteristic genetic fingerprints of nucleic acid substrates obtained by cleavage reactions CFLP in both clinical and research applications^TMThe law can be used Become. In this example, differently labeled isolates (two different dyes) were combined into one It shows that it can be cleaved in the tubing and analyzed using a fully automatic DNA sequencer.   An N-hydroxyl is added to the sense strand using PCR and primers labeled with a fluorescent dye. Sisuccinimidyl ester JOE-NHS (JOE) or FAM-NHS (F AM) was prepared. Bios on antisense strand Included tin label. The substrate used in this experiment was the exon of the tyrosinase gene. 15 from 4 wild-type (SEQ ID NO: 27) and 422 mutants (SEQ ID NO: 71) It was a 7 bp fragment.   The wild-type and 422 mutant tyrosinase gene substrates are Wild type (pcTYR-N1Try: Bouchard, B., et al., J. Exp. Med. 169: 2029, 1989). 422) (pcTYR-A422: Giebel, L.B., et al., 87: 1119, 1991). ) Was amplified from a cDNA plasmid clone containing any of the above. The following primers Each double-stranded substrate is amplified by PCR using a pair of Labeled with either dye or fluorescent dye. SEQ ID NO: 30 containing the 5 'biotin molecule Antisense primers are available from Integrated DNA Technologies, Inc. (IDT, Coralvil le, IA). Wild-type and biotinylated antisense primers The synthesis of both 422 mutant substrates was started. SEQ ID NO: 29 labeled with JOE Was used to initiate the synthesis of the wild-type tyrosinase gene. FA M using the sense primer of SEQ ID NO: 29 The synthesis of the enzyme gene was started. JOE and FAM labeled primers were Genset (Par is, France).   The PCR reaction was performed as follows. Each dNTP in 1X PCR buffer In a 100 μL reaction containing 50 μM and 1 μM of each primer of a defined primer pair With 1-2 ng of plasmid DNA from the wild type or 422 mutant Used as NA. Add Chill Out 14 to the reaction tube containing the above mixture.^TMLiquid wax (MJ (Research, Watertown, MA). Heat the reaction tube to 95 ° C for 1 minute, Next, it was cooled to 70 ° C. Next, Taq DNA polymerase (Perkin-Elmer) Added as 2.5 units of enzyme in 5 μL of 1 × PCR buffer. Tube at 95 ° C Heat for 45 seconds, cool at 50 ° C for 45 seconds, heat at 72 ° C for 1 minute and 15 seconds, repeat 35 cycles I returned. After the last cycle, the tubes were incubated at 72 ° C. for 5 minutes.   The PCR product was purified as follows. In buffer containing 0.5X TBE Were separated by electrophoresis on a 6% polyacrylamide gel (29: 1 cross-linking). D NA was visualized by ethidium bromide staining, and a 157 bp fragment was cut out from the gel. . 0.5M NH from DNA from gel slice_FourIn a solution containing OAc, 0.1% SDS and 0.1M EDTA Eluted overnight by passive diffusion. Next, 4 µg of glycogen carrier was added to the DNA. Precipitated with ethanol in the presence of. Pellet the DNA and add H_TwoO30μL Resuspended.   The cleavage reaction was performed as follows. H_TwoO Each double-stranded DNA in a total volume of 6 μL About 100 fmol of the substrate (1 to 3 μL of each gel-purified DNA) was added to 500 μL of thin-walled Into a centrifuge tube (Perkin-Elmer). Heat the tube to 95 ° C for 10 seconds to denature the substrate The tube was then quenched to 50 ° C. (this step allows intrastrand hydrogen between complementary bases). The bond is formed and takes on a unique secondary structure). 50mM MOPS (pH 7.2) 2μ L, 1mM MnCl_Two 1 μL and Cleavase^TMAdd 1 μL of BN (50 ng / μL) The cleavage reaction was started. Heat at 95 ° C for 10 seconds, and cool to 50 ° C Gram thermocycler (Perkin-Elmer DNA Thermal Cycler 480, Norwa lk, CT). The reaction was then incubated at 50 ° C. for 5 minutes, The reaction was stopped by adding 1 μL of 10 mM EDTA.   After the cleavage reaction, use the ABI 373A DNA Sequencer (Foster City, CA) for the sample. Gel electrophoresis. Prior to loading, 95% formamide and 10 mM EDTA The sample was denatured by adding 5 μL of a solution containing, and heated to 90 ° C. for 2 minutes. 1X T 6% polyacrylamide in BE buffer (89 mM Tris-borate, pH 8.3, 2 mM EDTA) 5 μL of the sample was separated by electrophoresis with Dogel (19: 1 cross-linking) and 6 M urea. . The gel was run at 30 watts for 14 hours. Sig obtained from four wavelength channels Na File from the Applied Biosystem Data Collection program on a Macintosh computer. Was collected using a system. The raw data is stored in the BaseFinder program (Giddings, M. et al ., Nucl. Acids Res. 21: 4530, 1993). Migration caused by the use of overlapping dyes and different dye labels. Was corrected.   The results are shown in FIG. FIG. 45 shows 2 corresponding to wild-type and mutant samples. Two lines representing one channel signal are shown. Wild-type sa labeled with JOE dye The sample is indicated by a thin line. Mutant sample labeled with FAM dye (R422Q ) Is indicated by a thick line. For comparison, a high-resolution polyacrylic acid containing separated cleavage products A photograph of a luamide gel (10% gel with a 19: 1 degree of crosslinking) is shown above the line ( The upper lane contains the cleavage fragment generated by cleavage of the wild-type substrate, the lower lane (Including cleavage fragments generated by cleavage of the R422Q mutant substrate). Sense strand The cleavage product shown in the gel containing a biotin label at the 5 'end of And visualized as described in Example 8a. Arrow 422 suddenly Selective bands seen on cleavage of mutant substrates from fully automated DNA sequencer Point to the corresponding peak of the prepared line (the arrows are 1 to 7 starting from the left side of FIG. 45). Is written).   Comparison of the two lines indicates cleavage of the wild-type and 422 mutant substrates in the same reaction. Differences in the cleavage pattern caused by this were detected by a fully automatic DNA sequencer. And For example, cleavage of the 422 substrate results in a cleavage product of about 56 nucleotides. However, this is not seen when the wild-type substrate is cleaved. Generating this 56 nucleotides The object is seen as the peak indicated by arrow 6 in FIG. FIG. 45 shows the mutant substrate Not only does cleavage yield new cleavage products, but wild-type Shows enhanced cleavage of certain structures compared to substrate (mutant compared to wild type) Compare the intensities of the peaks corresponding to arrows 2-5 in the variant line). further Certain cleavage products are commonly found on the two substrates, which are referred to as reference markers. (See arrows 1 and 7).   The above results demonstrate the characteristic genetic fingerprint of the nucleic acid substrate generated by the cleavage reaction. 2 shows that lint can be detected using a fully automatic DNA sequencer. This result It also shows that the cleavage reaction can be performed as multiple reactions. In this experiment , Both wild-type and mutant dsDNA substrates react in the same reaction (ie, multiple reactions). ) And then separated by the same electrophoresis using a fully automatic DNA sequencer.                                 Example 19         Cleavase^TMIdentification of virus strains using reactions   In the above embodiment, Cleavase^TMUsing human β-globin and To detect single base changes in fragments of various sizes derived from the tyrosinase gene. And These examples are useful for detecting and characterizing mutations in human populations. Cleavase^TMIt showed that the reaction could be used. Various virus strains Cleavase to detect sequence diversity characteristic of^TMReaction used Here's what you can do:   Simian immunodeficiency virus (SIV) infection in monkeys is a cause of human immunodeficiency in humans. It is commonly used as an animal model for the transmission of ills type 1 (HIV). SIV organisms The biological isolate includes multiple virus strains. Infect monkeys with biological isolates of SIV This gives a unique subset of virus stock from infected animals (specific strains). Can also infect tissue culture cells). Varies from infected animal by route of infection Virus genotype is isolated (Trivedi, P. et al., Journal of Virology 68 : 7649, 1994). SIV's Long Terminal Repeat (LTR) is a different virus Contains different sequences among different strains and is used as a marker to identify viral genotypes. Can be   Develop rapid methods to identify virus strains in samples (eg, clinical isolates) In vitro cloning using an uncloned biological SIV stock ro after infection of cultured cells or by intravenous or intrarectal inoculation. Cleavage against a characterized SIV genotype isolated after infecting Cleavase^TMThe reaction was used. In vitro feeling of CEMx174 cell line After staining (L. CEM / 251/12 clone), after intravenous inoculation of monkeys (L100. 8-1 clone), after low-dose inoculation of monkeys (L46.16-10 and L4 6.16-12 clones) and after high dose rectal inoculation of monkeys (L19.16-3). And L36.8-3 clone). The loan is C. David Pauza (Wisconsin Primate Research Center, Madison, WI) Obtained from. These clones are described in the literature (Trivedi, P. et al., Journal of Vir. ology 68: 7649, 1994). These plasmid clones Double-stranded DNA (dsDNA) containing the viral LTR sequence for the cleavage reaction ) Used for fabrication. A) Preparation of substrate DNA   Six SIV plasmids to create dsDNA substrates for the cleavage reaction Was used as a template for PCR. The primer pair used was U3-R of SIV LTR. It spans the border and amplifies a fragment of about 350 bp. In this part of the SIV LTR Is for transcription factor recognition sequences (including Sp1 and NF-KB) and for transcription initiation TATA box is contained and is polymorphic in various virus strains (Trivedi, P. et al., supra).   To amplify the SIV LTR clone by PCR, Different primer pairs were used. SEQ ID NO: 61 directs synthesis of the (+) chain of the SIV LTR Starting with 5'-GGCTGACAAGAAGGAAACTC-3 '. Arrangement Column no. 62 initiates the synthesis of the (-) chain of the SIV LTR, 5'-CCAGGGCG GCGGCTAGGAGAGATGGG-3 '. Opening of (+) chain of LTR In order to visualize the cleavage pattern generated by the cleavage, a biotin label was added to the 5 'end. A primer comprising SEQ ID NO: 61 and an unlabeled primer comprising SEQ ID NO: 62 PCR was performed using Caused by cleavage of the (-) chain of the viral LTR SEQ ID NO: 62 with a biotin label at the 5 'end to visualize the cleavage pattern And PCR using a pair of a primer consisting of Carried out.   The PCR reaction was performed as follows. Each dNTP is 60 μM, specified 0.2 μM of each primer of the primer pair, 10 mM Tris-Cl, pH 9.0 (25 ° C.), 2 mM MgCl_Two, 50 mM KCl, 0.1% Triton X-100 in 100 μL of the reaction solution 10 to 20 ng of plasmid DNA from each of the SIV LTR clones was Used as NA. Add 2 drops of light mineral oil to the reaction tube containing the above mixture And heat the tube at 96 ° C. for 3 minutes, then use Taq DNA polymerase (Perkin-Elm). er) with 20 mM Tris-HCl, pH 8.0, 0.5 μL, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% 2.5 units of enzyme in glycerol and 0.5% Tween 20 and 0.5% Nonidet P-40 Added as. Heat tube at 96 ° C for 45 seconds, cool at 60 ° C for 45 seconds, add at 72 ° C for 1 minute The heat was repeated for 35 cycles. After PCR, the reaction mixture is separated from the mineral oil. Release and add 5 μL of 5 M NaCl, 4 μL of 10 mg / ml glycogen and 10 μL 250 μL of 0% ethanol was added. After incubating at -20 ° C for 1 hour, DN Centrifuge A at maximum speed for 5 minutes in a Marathon Micro A centrifuge (Fisher Scientific). And pellet in 40 mM 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA TE. Resuspended.   The PCR product was gel purified as follows. Fill DNA with buffer (95% Lumamide, 5mM EDTA, 0.02% bromophenol blue, 0.02% xylene cyano G) mixed with 0.5 volume and heated at 75 ° C. for 2 minutes. The product is 7M urea, 0.5X TBE Using 6% polyacrylamide denatured gel (19: 1 cross-link) in buffer containing Separated by electrophoresis. Visualize the DNA with ethidium bromide staining and identify product bands. Cut out from the le. 0.5M NH from DNA from gel slice_FourOAc, 0.1% SDS and 0.1M It was eluted overnight in the solution containing EDTA by the dynamic diffusion method. Next, convert the DNA to tRNA Ethanol was precipitated in the presence of 4 μg of the carrier. Pellet DNA, 0.2M N The cells were resuspended in 50 μL of aCl, 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA. Etano with DNA And resuspended in 50 μL of TE. The final DNA concentration was determined for each double-stranded SIV   It was estimated to be 40 fmol / μL in the LTR PCR product. B) DNA sequence analysis of SIV LTR PCR product   The DNA sequences of the six PCR fragments prepared in A) above were converted according to the manufacturer's instructions. fmol^TMIt was determined using the DNA Sequencing System (Promega). For each sequencing reaction 0.2 pmol of PCR product and two 5'-biotinylated PCR primers One of SEQ ID NOs: 74 and 75 was used at 2 pmol (ie, the PCR fragment Were sequenced). After the sequencing reaction, the reaction determination product is electrophoresed. separated. After electrophoresis, a DNA buffer was prepared by the method described in Example 17 with the following modifications. Were transferred to a nylon membrane and visualized. TBS buffer (100 mM Tris-HCl, pH 7.4, 68 mM NaCl) 0.2% blocking reagent (Boehringe-Manheim) and Replace the solution containing 0.2% SDS with 1X Sequence Images Blocking Buffer (USB). I used it.   The sequence of a 351 bp fragment derived from the L100.8-1 LTR clone was sequenced. Reference numeral 63 indicates. 340 bp from the L46.16-10 LTR clone The sequence of the fragment is shown in SEQ ID NO: 64. L46.16-12 derived from LTR clone The sequence of the 340 bp fragment is shown in SEQ ID NO: 65. L19.16-3 LTR The sequence of the 351 bp fragment derived from the clone is shown in SEQ ID NO: 66. LCEM / The sequence of a 351 bp fragment derived from the 251/12 LTR clone is shown in SEQ ID NO: 6. FIG. L36.8-3 Sequence of a 351 bp fragment derived from LTR clone Is shown in SEQ ID NO: 68.   Analysis of the sequenced LTR fragment revealed the L100.8-1 LTR sequence (SEQ ID NO: 63) shows that they have multiple substitutions and deletions compared to 63). 6 LTRs The L100.8-1 LTR sequence was selected as a control for comparison between loans. I chose. For ease of discussion, the L100.8-1 LTR sequence (SEQ ID NO: 6) The nucleotide at the beginning or 5 'end of the (+) strand of 3) is defined as No. 1, The later or 3 'terminal nucleotide is defined as number 351.   FIG. 46 shows the nucleotide sequences of the six LTR clones. In the control clone A certain L100.8-1 (SEQ ID NO: 63) is shown in the upper row again. Sequences shown in bold are black Change in sequence when compared to the sequence of SEQ ID NO: L100.8-1 (SEQ ID NO: 63). You. The sequence shown in brackets in FIG. 46 is a very stable hair with a 14 base pair stem. The pin structure (12/14 bases in the stem are complementary) and control clone L100 . 8-1 (SEQ ID NO: 63) palindro capable of forming a 7 nucleotide loop This represents the sequence of the game. This hairpin structure is present in all six LTR clones, , Stem sequences and loop structures vary between clones.   The L46.16-10 sequence compared to the L100.8-1 sequence (SEQ ID NO: 63) (SEQ ID NO: 64) has seven substitutions and nucleotides 65-75 of SEQ ID NO: 63. It has one deletion consisting of the corresponding 11 nucleotides. The substitutions are as follows : C substituted by T at position 28 (C28T), C57T, G90A, C97T, G2 38A, G242A and G313A. L46.16-12 sequence (SEQ ID NO: 6) 5) is 7 substitutions and 11 nucleotides corresponding to nucleotides 65-75 of SEQ ID NO: 63. It has one deletion consisting of a nucleotide. The substitutions are as follows: C28T, C57T, G90A, C97T, A103G, G242A and G313A. L1 The 9.16-3 sequence (SEQ ID NO: 66) has two substitutions: A94C and A317T You. The LCEM / 251/12 sequence (SEQ ID NO: 67) has seven substitutions: G26A, G Has 72A, C97T, G258A, A281C, G313A and C316T You. The L36.8-3 sequence (SEQ ID NO: 68) has six substitutions: G60A, G172A. , G207A, G221A, T256C and C316T. C) Cleavage reaction conditions and CFLP of (−) chain of SIV LTR^TManalysis   A double-stranded substrate corresponding to the SIV LTR sequence shown in SEQ ID NOs: Labeling on the (-) strand using R and a primer pair corresponding to SEQ ID NOs: 61 and 62 Was. The primer of SEQ ID NO: 62 ((-) strand primer) has a biotin label at the 5 'end. It included intellect. Perform PCR and isolate reaction products as described in section A) did.   The cleavage reaction was performed as follows. About 60 fds of dsDNA substrate in 6 μL of water The reaction tube was made up of The following reagents were added to the DNA: 5X CFLP with 150 mM KCl^TM 2 μL of buffer (pH 7.2) (giving a final concentration of 30 mM KCl) and Cleavase (Cleavase) ase)^TMBN enzyme (25 ng in 1X dilution buffer). Cleavase^TMInstead of BN enzyme And H_TwoExcept for using 1 μL of O, the reaction tube containing all the above components was uncut or An enzyme-free control was performed. PTC-100 reaction tube^TMProgrammable Thermal Contro The DNA was denatured by heating in an ller (MJ Research. Inc.) at 95 for 10 seconds. Modification Next, the tube was quenched to 40 ° C. 2mM MnCl_Two1 μL (to give a final concentration of 0.2 mM) In addition, the cleavage reaction started immediately. The tubes were incubated at 40 ° C. for 5 minutes. Stop The reaction was stopped by adding 6 μL of buffer. Heat the sample at 85 ° C for 30 seconds. 5 μL of the reaction was applied to a 12% polyacrylamide gel (19: 1 cross-link) and 0.5X Separation was achieved by electrophoresis using 7M urea in buffer containing TBE.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8a. I put the membrane. The DNA was transferred to a membrane, the membrane was dried, and 0.2% blocking reagent (Boeh ringer Manheim): Wash in 0.2% SDS in 100 mM Tris-HCl, pH 7.4, 68 mM NaCl , Treated with 1X SAAP buffer, CDP-Star^TMExample 8a by reacting with (Tropix) Exposure to X-ray film as described under. Figure 4 shows the resulting autoradiograph. FIG.   FIG. 47 shows the cleavage pattern corresponding to cleavage of the (−) strand of the double-stranded LTR substrate. . In FIG. 47, the lane indicated by “M” is a molecular weight marker (as described in Example 8). Prepared). Lanes 1-6 are L100.8-1, L46.16-10, L 46.16-12, L19.16-3, LCEM / 251/12 and L36.8. -3 shows cleavage products generated by cleavage of the LTR PCR fragment. Les Regions 7-12 are those of the six LTR substrates in the same order as described in Lanes 1-6. Includes each uncut control.   The results shown in FIG. 47 show the cleavage or CFLP corresponding to each LTR substrate.^TMThe pattern is Size from about 350 nucleotides (uncleaved substrate) to less than 24 nucleotides Indicates that a plurality of bands are included. A bar located less than about 100 nucleotides in length Are different among the six clones, indicating that the different SIV LTR isolates Means a nucleotide change characteristic of CFLP^TMTo consider patterns Thus, five unique cleavage patterns in six SIV LTR isolates It became clear that it was detected. From the DNA sequence data, six LTR clones Everything turned out to be unique. However, CFLP^TMThe pattern is The clones shown in Regions 2 and 3 appeared to be the same.   CFLP generated by cleavage of the (-) chain of all six substrates^TMThe pattern is about length Includes a strong band corresponding to a 100 nucleotide fragment. This band is the (-) chain 6 in the long palindromic sequence located 97 nucleotides from the 5 'end of Corresponds to cleavage of all three LTR substrates (see region in brackets in FIG. 46) . This palindromic sequence forms a very stable hairpin structure in single-stranded DNA And Cleavase^TMProvides optimal substrates for BN enzymes. this Cleavage of the hairpin structure is expected to produce a fragment of about 100 nucleotides.   L46.16-10 which is the LTR substrate shown in lanes 2 and 3 (SEQ ID NO: 64) And L46.16-12 (SEQ ID NO: 65) were produced from the same animal using the same route of infection. (Trivedi, P. et al., Supra). These substrates are detectable Region other than a single base in the region corresponding to the cleavage site (ie, 100 nucleotides or less) With the same sequence: L46.16-10 clone (SEQ ID NO: 64) has SEQ ID NO: 63 G is changed to A at position 239 as compared to the control sequence shown in (G239A). By examining the DNA sequences of these two clones, this substitution was It is evident that it is located in the loop region of the pinned structure (in parentheses in FIG. 46). See palindrome region enclosed). A single base between these two sequences Is located in the loop region of the hairpin structure. Then different CFLP^TMDNA secondary of two substrates enough to generate a pattern The structure may not have changed. Reactions that destabilize strong hairpin structures Detect single base differences between these two clones by changing conditions Will be able to do that.   The result shown in FIG.^TMSix SIV L used in the study using the reaction Most (5/6 or 83%) of sequence diversity present in TR clones can be detected Indicates that Further, FIG.^TMReaction seeks secondary structure of single-stranded nucleic acid It is a sensitive means for D) SIV LTR (+) chain cleavage reaction conditions and CFLP^TManalysis   A double-stranded substrate corresponding to the SIV LTR sequence shown in SEQ ID NOs: 76-81 was synthesized using PC R and the primer pair corresponding to SEQ ID NOs: 74 and 75 on the (+) strand Labeled. The primer of SEQ ID NO: 61 ((+) strand primer) has a bio Included tin label. Perform PCR as described in section A) and generate the reaction. Was isolated. Cleavage reaction, electrophoresis and DNA visualization as described in section C) Was performed. The resulting autoradiograph is shown in FIG.   FIG. 48 shows the results corresponding to the cleavage products of the (+) strand of the six SIV LTR fragments. The following shows the pattern. Lanes indicated by "M" are molecular weight markers (described in Example 8). Prepared as described above). Lanes 1-6 are L100.8-1, L46.16- 10, L46.16-12, L19.16-3, LCEM / 251/12 and L 36.8-3 The cleavage products generated by cleavage of the LTR PCR fragment Show. Lanes 7-12 show six LTR groups in the same order as described in lanes 1-6. Includes each uncut control of quality.   As indicated by cleavage of the (-) chain of the LTR clone, the SIV LTR substrate CFLP corresponding to each (+) chain^TMPatterns make most of a particular nucleotide substitution Includes unique properties that characterize it. For example, L46.16-10 (SEQ ID NO: 64) L46.16-12 (SEQ ID NO: 65) (lanes 2 and 3 in FIG. 48) has 11 nucleotides. Otide deletions can be easily detected. This deletion is one of the three Sp1 binding sites. Animals that have been removed and infected by low-dose rectal inoculation of the virus stock And characteristic changes in the predominant SIV genotype (Trivedi, P. et al., Supra). . (+) Of substrate from clones L46.16-10 and L46.16-12 CFLP generated by chain cleavage^TMThe pattern is the same under these reaction conditions. Was.   The above results indicate that CFLP^TMThe different strains (ie genotype) of the virus quickly It can be used as an effective means of identification. Virus or other The ability to rapidly identify specific strains of pathogenic organisms is clinically important. The above results Is CFLP^TMTo provide a method for rapidly identifying strains or species using reactions. Is shown.                                 Example 20         Effect of changing salt concentration on cleavage reaction using single-stranded DNA substrate   In Example 11, when single-stranded DNA was used as a substrate, cleavedase ( Cleavase)^TMShows that the reaction is not sensitive to large changes in reaction conditions Was done. In Example 11, the cleavage reaction was performed over a wide range of salt concentrations (0-50 mM KCl) and single-stranded It has been shown that this can be performed by using in combination with a substrate. In this embodiment Shows the effect of using other salts instead of KCl on cleavage using single-stranded DNA substrate. The reaction was examined. A) Effect of using NaCl instead of KCl in a cleavage reaction using a single-stranded template   The use of NaCl instead of KCl allows 5'nuclei on single-stranded DNA substrates. To examine the effect on the cleavage pattern created by creatase activity, The following experiment was performed. 1mM MOPS, pH8.2, 1mM MnCl_TwoAnd various amounts of NaCl A fixed amount of Cleavase in the impulse^TMOne casting in the presence of BN enzyme (50ng) The mold was incubated.   157 nucleotide fragment from sense strand of exon 4 of tyrosinase gene (SEQ ID NO: 47: Prepared by the method of Example 8b) Approximately 100 fmoles were In a wall microcentrifuge tube (Perkin-Elmer, Norwalk, CT), 1X CFLP^TMBuffer, pH 8.2 And 1.33 mM MnCl_Two(Final concentration 1mM MnCl_TwoIn a volume of 15 μL. Last NaCl was added to a concentration of 0, 10, 20, 30, 40, 50, 75 or 100 mM. Final The reaction volume was 20 μL.   10mM MOPS, pH8.2, 1mM MnCl_TwoAnd tubes containing 100 fmoles of substrate DNA salt-free, Used as a control without enzyme (Cleavase)^TMSterilized steam instead of BN Using distilled water, all reaction components were added before denaturation at 95 ° C).   The tube was heated at 95 ° C for 20 seconds and then quenched to 65 ° C. MnCl_Two10mM MO not including PS, Cleavase in pH 8.2^TMDilute enzyme mixture containing 1 μL of BN [1 X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 50 ng / μL in μg / ml BSA). Started.   After 5 minutes at 65 ° C., stop buffer (95% formamide, 10 mM EDTA, 0.05% bromide) (Phenol blue, 0.05% xylene cyanol) was added to stop the reaction. . The sample was heated at 72 ° C. for 2 minutes and each reactant was treated as described in Example 8a. 7 μL was applied to 10% polyacrylamide gel (19: 1 cross-linking) and 0.5 × TBE Were separated by electrophoresis using 7M urea in a buffer containing   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8a. I put the membrane. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline 1X I-Block (Tropi) conjugated with phosphatase (United States Biochemical) x, Bedford, MA), washed, and CDP- as described in Example 8a. Reaction with StarTM (Tropix, Bedford, MA). However, membrane 1cm^Two0.01ml CD per P-StarTM was added. The membrane was exposed to X-ray film as described in Example 8a. FIG. 49 shows the obtained result.   In FIG. 49, the lane indicated by “M” is a molecular weight marker (as described in Example 8a). Prepared in this manner). Lane 1 contains a salt-free, enzyme-free control and was cleaved The figure shows the mobility of the template DNA not present. Lanes 2 to 9 are 0, 10, 20, 30 respectively Cleavase in a buffer containing 50, 40, 50, 75 or 100 mM NaCl^{T M} Includes reaction products incubated in the presence of BN enzyme.   The results shown in FIG. 49 indicate that the use of NaCl instead of KCl Cleavage pattern generated using tyrosinase DNA substrate of leotide (SEQ ID NO: 34) Has little or no effect on the application. KCl (Example) 13b, see FIG. 32) or NaCl (FIG. 49). With the NA substrate, the cleavage pattern depends essentially on the salt concentration Was observed. B) (NH) instead of KCl in the cleavage reaction using a single-stranded template_Four)_TwoSO_FourFor Effect   In a similar manner as described in section A) above, (NH) may be substituted for KCl._Four)_TwoSO_FourTo Using 5 'nuclease activity on a single-stranded DNA substrate The effect on the cleavage pattern was examined. The cleavage reaction is exactly as described in section A). Same but with different amounts of (NH_Four)_TwoSO_FourWas used.   A 157 nucleotide fragment from the sense strand of exon 4 of the tyrosinase gene ( SEQ ID NO: 47: Prepared as in Example 8a) Approximately 100 fmoles in 500 μL of thin wall 1X CFLP in a microcentrifuge tube (Perkin-Elmer, Norwalk, CT)^TMBuffer solution, pH 8.2 1.33mM MnCl_Two(Final concentration 1mM MnCl_TwoIn a volume of 15 μL. Final darkness So that the degree is 0, 10, 20, 30, 40, 50, 75 or 100 mM (NH_Four)_TwoSO_FourAdd Was. The final reaction volume was 20 μL.   10mM MOPS, pH8.2, 1mM MnCl_TwoAnd tubes containing 100 fmoles of substrate DNA salt-free, Used as a control without enzyme (Cleavase)^TMSterilized steam instead of BN Using distilled water, all reaction components were added before denaturation at 95 ° C).   The tube was heated at 95 ° C for 20 seconds and then quenched to 65 ° C. MnCl_Two10mM MO not including PS, Cleavase in pH 8.2^TMDilute enzyme mixture containing 1 μL of BN [1 X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 The cleavage reaction is started by the immediate addition of 5 μL of 50 ng / ml in 50 mg / ml BSA). Was.   After 5 minutes at 65 ° C., the reaction was stopped by adding 16 μL of stop buffer. 72 samples Heat at 2 ° C. for 2 minutes and add 7 μL of each reaction to 10% as described in Example 8a. Polyacrylamide gel (19: 1 cross-linking) and 0.5X TBE in buffer Separated by electrophoresis using 7M urea.   After electrophoresis, the DNA was transferred to a membrane and detected as described above in section A). Profit The obtained autoradiograph is shown in FIG.   In FIG. 50, the lane indicated by “M” indicates the molecular weight marker described in Example 10a. Including. Lane 1 contains the enzyme-free control and shows the mobility of the uncleaved template DNA. Is shown. Lanes 2 to 9 show 0, 10, 20, 30, 40, 50, 75 or 100 mM, respectively ( NH_Four)_TwoSO_FourIn a buffer containing Cleavase^TMPresence of BN enzyme Includes reaction products incubated below.   The result shown in FIG._Four)_TwoSO_FourCleavage pattern is severe due to the presence of Indicates that it was inhibited. The reaction was performed at 20 mM (NH_Four)_TwoSO_FourEven a small amount is perfect 10 mM (NH_Four)_TwoSO_FourThe extent of the cleavage reaction in 50 mM K Cl or NaCl, comparable to that obtained with 0 mM (NH_Four)_TwoSO_Fourso It was significantly lower than that obtained. However, 10 mM (NH_Four )_TwoSO_FourThe cleavage pattern obtained in (1) is a 157 nucleotide template (SEQ ID NO: 34). ) To (NH_Four)_TwoSO_FourOr when incubated in the absence of KCl or NaCl This is the same as the cleavage pattern obtained in this case. This is because the choice of salts involved in the cleavage reaction is , Cleavase^TMOf the site as a substrate recognized by the BN enzyme Indicates no effect on the properties (ie, the observed inhibitory effect is (NH_Four)_TwoSO_FourEffect on the enzymatic activity and on the cleavage structure Absent). C) Effect of increasing KCl concentration on cleavage of single-stranded substrate   The effect of increasing the KCl concentration in the cleavage reaction using the single-stranded DNA The test was performed by performing a cleavage reaction at a KCl concentration in the range of 00 mM. Anti-cleavage The reaction was carried out as described in section A), but with 0, 25, 50, 75 or KCl was added to give a final concentration of 100 mM and 200 fmol of substrate was used. The substrate DNA was denatured by incubating at 95 ° C for 5 seconds.   After the cleavage reaction, the sample is subjected to electrophoresis as described above in section A), Transferred and detected. The resulting autoradiograph is shown in FIG.   In FIG. 51, the lane indicated by “M” contains the molecular weight marker described in Example 8a. No. Lane 1 is an enzyme-free control, and lanes 2-7 are 0, 25, and 50, respectively. , 75, 100 or 100 mM KCl (100 mM sample is repeated twice) ) In the presence of   The results shown in FIG. 51 show that the degree of cleavage of the cleavage reaction was low as a function of increasing KCl concentration. (Although detectable cleavage remained even at 100 mM KCl) ). In addition, the single-stranded substrate is converted to Cleavase.^TMCleavage with BN enzyme The fragment pattern generated by the reaction is affected by the KCl concentration present during the reaction. I can't. D) Effect of high concentration KCl on cleavage reaction using single-stranded substrate   Cleavase with relatively high concentration of KCl^TMThe ability of the reaction is 1 By performing the cleavage reaction in the presence of various concentrations of KCl above 00 mM Tested. C) Derived from exon 4 of the tyrosinase gene as described above in section The reaction was performed using 157 nucleotides (SEQ ID NO: 34), KCl was added to give a final concentration of 50, 200, 250 or 300 mM .   After the cleavage reaction, the sample is subjected to electrophoresis as described above in section A), Transferred and detected. The results (data not shown) show that at KCl concentrations above 100 mM It shows that the cleavage reaction is severely inhibited. However, up to 300 mM KCl Even with increasing salt concentration, some cleavage remained. E) Effect of KCl on stability of cleavage pattern at long incubation times Effect of concentration   The above results are for Cleavase^TMThe reaction is highly concentrated (ie, 50 m M or more) is inhibited by KCl or NaCl. This inhibition responds Does this effectively affect the stability of the cleavage pattern after extended time (ie, (For inhibition of enzyme activity) at 0 and 50 mM KCl The reaction was tested for an extended time.   A 157 nucleotide fragment from the sense strand of exon 4 of the tyrosinase gene ( SEQ ID NO: 47: Prepared by the method of Example 10a) Approximately 100 fmoles In a wall microcentrifuge tube (BioRad, Richmond, CA), 1X CFLP^TMBuffer solution, pH 8.2, 1 .33mM MnCl_Two(Final concentration 1mM MnCl_TwoAnd KCl to give a final concentration of 0 or 50 mM Was put. Final reaction volume was 20 μL.   For each time point tested, a control reaction without enzyme was prepared in parallel. this These enzyme-free controls were prepared as described above. However, Cleavase (Cleava se)^TMUse sterile distilled water instead of BN and add all reaction components prior to denaturation at 95 ° C. I got it.   The tube was heated at 95 ° C for 20 seconds and then quenched to 65 ° C. MnCl_Two10mM MO not including PS, Cleavase in pH 8.2^TMDilute enzyme mixture containing 1 μL of BN [1 X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 cleavage reaction by immediately adding 5 μL of 50 ng / ml in 50 μg / ml BSA) did. After enzyme addition, add Chill Out 14 to each tube.^TM(MJ Research, Watertown, MA) 20μ L was added. The reaction was carried out at 65 ° C. for 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17 hours. Time went.   At the desired time, the reaction was stopped by adding 16 μL of stop buffer. Sample at 72 ° C And 7 μL of each reaction as described in Example 8a. Liacrylamide gel (19: 1 cross-linking) and 7 in a buffer containing 0.5X TBE Separated by electrophoresis using M urea.   After electrophoresis, the DNA was transferred to a membrane and detected as described above in section A). Profit The obtained autoradiograph is shown in FIG.   In FIG. 52, the lane indicated by “M” indicates the molecular weight marker described in Example 10a. Including. Lanes 1-10 contain the reaction products performed in the absence of KCl and 1-20 contain the reaction products performed in the presence of 50 mM KCl. Lane 1, 3, 5, 7 and 9 are 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17:00 Includes enzyme-free control incubated for 10 min. Lanes 2, 4, 6, 8, and 10 Incubate at 65 ° C for 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17 hours, respectively. Includes baked reaction products. Lanes 11, 13, 15, 17, and 19 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 17 hours, respectively, in 50 mM KCl Includes enzyme-free control incubated with. Lanes 12, 14, 16, 18 and 20, 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 17 hours at 65 ° C. , CFLP incubated in 50 mM KCl^TMContains the reaction products of the reaction.   The results indicate that cleavage is delayed in the presence of 50 mM KCl, Significant stabilization of the cleavage pattern was obtained (i.e., the rate of cleavage was dramatically reduced). And the large cleaved fragment was not cleaved into smaller fragments, Over time, the cleavage pattern remained the same). Incubation time Longer, reactions performed in the absence of KCl were significantly overdigested and at 65 ° C. After time, virtually no large fragments remain and considerable accumulation of small cleavage products I do. In contrast, reactions performed at 50 mM KCl for 30 minutes to 4 hours Is substantially stable between the two, and overdigestion is observed only at the longest time points, to the extent Not as much as observed in the absence of KCl.                                 Example 21     Comparison of cleavage patterns generated by cleavage of single-stranded and double-stranded DNA substrates   Cleavase^TMBN-mediated double-stranded DNA substrate In immersion-independent cleavage, DNA double strands are separated during the denaturation step before the addition of the enzyme. Separated. Therefore, the pattern generated by cleavage of the double-stranded template is It should be the same as the pattern caused by the crack. This estimation is based on the following experiment. I confirmed.   A 157 nucleotide fragment derived from the sense strand of exon 4 of the tyrosinase gene The single-stranded substrate (SEQ ID NO: 34) was prepared by the method described in Example 10b with the following modifications: Prepared. After gel purification and precipitation in the presence of a glycogen carrier, the PCR product is Resuspend in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and add 2M NH_FourOAc and Reprecipitated with 2.5 volumes of ethanol. Next, resuspend the DNA in 400 μL of TE did.   About 50 or 100 fmol of a single-stranded 157 nucleotide fragment (SEQ ID NO: 47) 1X CFLP in a 200 μL centrifuge tube (BioRad, Richmond, CA).^TMBuffer, pH 8.2 , And 1.33 mM MnCl_Two(Final concentration 1mM MnCl_TwoIn a volume of 15 μL. Final reaction volume was 20 μL. 20 μL of salt-free, enzyme-free control in parallel Prepared. These control reactions were Cleavase^TMInstead of BN enzyme And sterile distilled water, and all reaction components were added prior to denaturation at 95 ° C.   The reaction tube was heated at 95 ° C. for 5 seconds and then quenched to 65 ° C. MnCl_Two10mM not including Cleavase in MOPS, pH 8.2^TMDilute enzyme mixture containing 1 μL of BN [ 1X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 50 ng / μL in 0 μg / ml BSA) by immediately adding 5 μL. Started. After 5 minutes at 65 ° C., the reaction was stopped by adding 16 μL of stop buffer.   In the same experiment, a double-stranded form of a 157-nucleotide substrate was cleaved from Cleavase. )^TMCleavage with BN. This double-stranded substrate (SEQ ID NO: 27) was implemented with the following modifications: Prepared as described in Example 8b. Gel purification and precipitation in the presence of glycogen carrier Thereafter, the PCR product was resuspended in TE (10 mM ris-HCl, pH 8.0, 1 mM EDTA) and M NH_FourReprecipitated with OAc and 2.5 volumes of ethanol. Next, the DNA is TE Resuspended in 400 μL.   About 33 or 66 fmol of the double-stranded 157 bp fragment (SEQ ID NO: 27) was L in a thin-walled microcentrifuge tube (BioRad, Richmond, CA). 15 sterile distilled water μL was added.   The reaction tube was heated at 95 ° C. for 5 seconds and then quenched to 65 ° C. 10mM MOPS, pH 8.2, 0. 8mM MnCl_Two(Final concentration 10 mM MOPS, pH 8.2 and 0.2 mM MnCl_TwoDiluent yeast containing 5 μL of elemental mixture and Cleavase^TMBN [1X dilution buffer (0.5% NP40 , 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) [μL] The cleavage reaction was started immediately by adding 0.5 μL. Salt free, enzyme 20 μL of a double-stranded substrate control without any was prepared in parallel. Cleavage to this reaction Ze (Cleavase)^TMContains sterile distilled water instead of BN enzyme.   After 5 minutes at 65 ° C., the reaction was stopped by adding 16 μL of stop buffer. The sample was then heated at 72 ° C. for 2 minutes and the reaction product was prepared as described in Example 8a. Contains 10% polyacrylamide gel (19: 1 cross-linking) and 0.5X TBE Separation was achieved by electrophoresis using 7M urea in buffer.   After electrophoresis, the gel plate was separated and analyzed on a Nylon gel as described in Example 10a. The film was put on. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline 1-X I-Block (Tro) conjugated with a reactive phosphatase (United States Biochemical) pix, Bedford, MA), washed, and as described in Example 20a. CDP-Star^TM(Tropix, Bedford, MA) and exposed the film to X-ray film . FIG. 53 shows the obtained result.   In FIG. 53, lanes 1-3 contain reaction products from reactions involving single-stranded substrates. , Lanes 4-7 contain the reaction products from the reaction involving the double-stranded substrate. Lane 1 and And 3 are reactions from cleavage reactions containing 50 or 100 fmol of single-stranded substrate, respectively. Contains 7.0 μL of product. Lane 2 shows 7.0 μL of uncleaved single-stranded substrate control reaction. including. Lanes 4 and 6 show 33 and 66 fmoles of uncleaved double-stranded substrate, respectively. Contains 7.0 μL of the control reaction. Lanes 5 and 7 show 33 or 66 fmoles, respectively. Contains 7.0 μL of reaction product from a cleavage reaction involving a double-stranded substrate. Uncut double strand Note that the control is shown as a double line below the main band containing the 157 bp substrate. I want to be reminded. This doublet has different mobilities rather than degradation products It is believed to represent another structure that moves. This doublet was purified from denaturing gel It does not appear in experiments using double-stranded DNA (see Example 22).   Comparison of the cleavage patterns generated by cleavage of single-stranded or double-stranded substrates indicates that It was shown that the same pattern occurred.                                 Example 22       Cleavase using double-stranded DNA template^TMThe reaction is                   Sensitive to large changes in reaction conditions   The results shown in Example 11 indicate that Cleavase^TMReaction is MnCl_TwoPassing And KCl concentration, temperature, incubation time, vase)^TMFor significant changes in many reaction conditions, including the amount of BN enzyme and the DNA preparation, And was relatively insensitive. The results shown in Example 11 show that the single-stranded substrate Using Cleavase^TMWhen performing the reaction, This shows that the reaction is extremely stable. In the experiments described below, double-stranded substrate This indicates that the cleavage is limited to a rather narrow range of reaction conditions. A) Preparation of a double-stranded 157 bp fragment of exon 4 of the tyrosinase gene   In the following experiments, the double-stranded DNA template was converted to Cleavase.^TMFor reaction Test the effect of changing reaction conditions when The double-stranded substrate used is wild 157 bp fragment of the tyrosinase gene (SEQ ID NO: 27). This 157 The bp fragment was generated using the symmetric PCR described in Example 8b. Briefly state And about 75 fmoles of double-stranded substrate DNA were combined with primer 5 'in 1X PCR buffer. Biotin-GCCTTATTTTACTTTAAAAAT-3 '(SEQ ID NO: 32 ) 50 pmol, primer 5 'fluorescein-TAAAGTTTTGTGTT 50 pmol of ATCTCA-3 '(SEQ ID NO: 33), and 50 mM of each dNTP. Incubated together. A tube containing 95 μL of the above mixture was heated at 95 ° C. for 5 seconds. , Cooled to 70 ° C. Taq DNA polymerase in IX PCR buffer 5 μL of the enzyme mixture containing 25 units was added. Chill Out 14 for each tube^TM(MJ Res earch, Watertown, MA) was added.   The tube was heated at 95 ° C for 45 seconds, cooled at 50 ° C for 45 seconds, and heated at 72 ° C for 75 seconds. Was repeated for 30 cycles and incubated at 72 ° C. for 5 minutes after the last cycle. Was. The reaction was then ethanol precipitated to reduce the volume of gel purification. NaC 1 to a final concentration of 400 mM and glycogen (in distilled water) to a final concentration of 20 mM. Added to 0 μg / ml. Add 2.5 volumes of 100% ethanol to each tube. Cooled at -70 ° C for 2.5 hours. Pellet DNA and sterilize 1/5 volume of original Resuspended in distilled water.   The PCR product was gel purified as follows. Add an equal volume of stop buffer to each tube In addition, the tube was heated at 72 ° C. for 2 minutes. The product was subjected to a 6% denaturing polyacrylamide gel ( 19: 1 cross-linking) and 7 mM in a buffer containing 45 mM Tris-boric acid, pH 8.3 and 1.4 mM EDTA. Separation was performed by electrophoresis using M urea. Visualize the DNA with ethidium bromide staining, A 157 bp fragment was excised from the gel. Gels as described in Example 8a DNA was eluted from the rice by passive diffusion. However, diffusion takes place at room temperature for 2 days I let you. The DNA was then added in the presence of 200 mM NaCl without adding carrier molecules. Settled. The DNA was pelleted and resuspended in 150 μL TE. B) Effect of KCl concentration on double-strand cleavage reaction   To examine the effect of salt concentration on the cleavage reaction when using double-stranded substrates, 10mM MOPS, pH7.5, 0.2mM MnCl_TwoAnd various buffers containing 0-100 mM KCl Medium fixed amount of Cleavase^TMOne substrate in the presence of BN enzyme (25ng) Incubated.   A 157 bp fragment from exon 4 of the tyrosinase gene (SEQ ID NO: 27: A) Approximately 100 fmol was prepared in a 200 μL thin-walled microcentrifuge tube (BioR). ad, Richmond, CA) in 6.25 μL of sterile distilled water (final reaction volume 10 μL) I put it in. The tube was heated at 95 ° C for 15 seconds and quenched to 45 ° C. 2.7X CFLP^TMBuffer Solution, pH 7.5 (to give a final concentration of 1X), 0.53 mM MnCl_Two(Giving a final concentration of 0.2 mM), Cleavase^TMBN [1X dilution buffer (0.5% NP40, 0.5% Tween20, 20 mM 50 ng / μL in Tris-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) 5 μL and final concentration Enzyme mixture 3 containing KCl giving 0, 2.5, 5, 10, 15, 20, 25, 30, 50 or 100 mM The cleavage reaction was started by adding .75 μL. Final reaction volume is 10 μL there were. The enzyme solution was at room temperature before adding to the cleavage reaction. Contains no enzymes An uncut) control was prepared in parallel with 0 or 100 mM KCl, which was cleaved Cleavase)^TMThe difference was that sterile distilled water was used instead of BN.   After 5 minutes at 45 ° C., the reaction was stopped by adding 8 μL of stop buffer. 72 samples 2 minutes at 4 ° C. and 4 μL of each reaction was loaded on a 10% polyacrylamide gel (19: 1 crosslink) and 7M urea in buffer containing 0.5X TBE. And separated.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8a. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 54 shows the obtained autoradiograph. Shown in   In FIG. 54, the lane indicated by "M" contains a molecular weight marker. Lane 1 is 0 mM KC 1 shows the mobility of uncleaved template DNA, including the uncleaved control in l. Lane 2-1 1 is a buffer containing 0, 2.5, 5, 10, 15, 20, 25, 30, 50 or 100 mM KCl, respectively. Inside, Cleavase^TMIncubate the substrate in the presence of the BN enzyme And the reaction products generated by the reaction. Lane 12 shows the ink in a buffer containing 100 mM KCl. Includes an uncut control that has been developed.   The results shown in FIG. 54 show the results of Cleavase performed on the double-stranded DNA template. )^TMThe reaction was shown to be sensitive to changes in salt concentration. Containing more than 30 mM KCl Upon reaction, substantially no cleavage was detected. 157 bp tyrosinase DNA substrate (SEQ ID NO: 27) was replaced with Cleavase.^TMIncubate with BN enzyme In some cases, the same cleavage pattern was obtained even when the KCl concentration was varied from 0 to 30 mM. C) Effect of NaCl concentration on double-strand cleavage reaction   The use of NaCl instead of KCl for the 5'nucleic acid on double-stranded DNA substrates The effect on the cleavage pattern created by creatase activity was tested. Firshi 157 bp fragment from exon 4 of the enzyme gene (SEQ ID NO: 40: Example 22a Approximately 100 fmol was prepared in a 200 μL thin-wall microcentrifuge tube (BioRad , Richmond, CA) in 6.25 μL of sterile distilled water (final reaction volume 10 μL). And heated at 95 ° C. for 15 seconds. The tube was quenched to 45 ° C. 2.7X CFLP^TMBuffer, p H7.5 (giving a final concentration of 1X), 0.53 mM MnCl_Two(Giving a final concentration of 0.2 mM), cleave Alease (Cleavase)^TMBN [1X dilution buffer (0.5% NP40, 0.5% Tween20, 20 mM Tris -HCl, pH8.0, 50 mM KCl, 50 ng / μL in 10 μg / ml BSA)] 0.5 μL and final concentration 0 Enzyme mixture with NaCl to give 2.5, 5, 10, 15, 20, 25, 30, 50 or 100 mM 3.7 The cleavage reaction was started by adding 5 μL. Final reaction volume is 10 μL. Was. An enzyme-free (ie, uncleaved) control was prepared in parallel with 0 or 100 mM NaCl. , This is Cleavase^TMThe difference is that sterile distilled water is used instead of BN. I was   After incubating the reaction at 45 ° C. for 5 minutes, 8 μL of stop buffer was added to stop the reaction. Stopped. The sample was heated at 72 ° C. for 2 minutes and 4 μL of each reaction was added to 10% 7M urine in a buffer containing acrylamide gel (19: 1 cross-linking) and 0.5X TBE Separation was carried out by electrophoresis using element.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 55 shows the obtained autoradiograph. Shown in   In FIG. 55, the lane indicated by "M" contains a molecular weight marker. Lane 1 contains 0 mM Na An uncleaved template DN containing an enzyme-free control incubated in a buffer containing Cl A shows the mobility of A. Lanes 2-11 are 0, 2.5, 5, 10, 15, 20, 25, 3 respectively. Cleavase in a buffer containing 0, 50 or 100 mM NaCl^TMBN enzyme Reaction product resulting from cleavage of a 157 bp substrate (SEQ ID NO: 27) in the presence of No. Lane 12 shows enzyme-free pairs incubated in a buffer containing 100 mM NaCl. Including light.   The results shown in FIG. 55 show the results for Cleavase performed on the double-stranded DNA template. )^TMThe reaction was shown to be sensitive to changes in NaCl concentration. Actual above 20 mM NaCl No cleavage was qualitatively detected. 157 bp tyrosinase DNA template (SEQ ID NO: No. 27) Cleavase^TMWhen incubating with BN enzyme, The same cleavage pattern was obtained even when the concentration of NaCl varied from 0 to 20 mM. D) (NH) instead of KCl in the cleavage of the double-stranded template_Four)_TwoSO_FourThe effect of using   In a similar manner as described in Example 20b, the cleavage reaction when using a double-stranded substrate In response to (NH) instead of KCl_Four)_TwoSO_FourTested that can be used . The cleavage reaction was exactly the same as described in Examples 22b and c, but with KCl or Various amounts of (NH_Four)_TwoSO_FourWas used.   A 157 bp fragment from exon 4 of the tyrosinase gene (SEQ ID NO: 40: A) Approximately 100 fmol) were prepared in a 200 μL thin-walled microcentrifuge tube (prepared as described in 6.25 μL of sterile distilled water (final reaction volume 10 μL) in BioRad, Richmond, CA ) And heated at 95 ° C. for 15 seconds. The tube was quenched to 45 ° C.   2.7X CFLP^TMBuffer, pH 7.5 (to give a final concentration of 1X), 0.53 mM MnCl_Two(Final concentration 0.2 mM), Cleavase^TMBN [1X dilution buffer (0.5% NP40, 0.5% % Tween 20, 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 50 μg / ml in 10 μg / ml BSA] Give 0.5 μL and final concentration 0, 2.5, 5, 10, 15, 20, 25, 30, 50 or 100 mM (NH_Four )_TwoSO_FourThe cleavage reaction was started by adding 3.75 μL of an enzyme mixture containing Most The final reaction volume was 10 μL. 0 or 100 controls without enzyme (ie, uncleaved) mM (NH_Four)_TwoSO_FourPrepare in parallel with this, this is Cleavase^TMInstead of BN Using sterile distilled water.   After incubating the reaction at 45 ° C. for 5 minutes, 8 μL of stop buffer was added to stop the reaction. Stopped. The sample was heated at 72 ° C. for 2 minutes and 4 μL of each reaction was added to 10% 7M urine in a buffer containing acrylamide gel (19: 1 cross-linking) and 0.5X TBE Separation was carried out by electrophoresis using element.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 56 shows the obtained autoradiograph. Shown in   In FIG. 56, the lane indicated by "M" contains a molecular weight marker. Lane 1 shows 0 mM (NH_Four )_TwoSO_FourUncleaved substrate, including no enzyme control, incubated in buffer containing Shows the mobility of DNA. Lanes 2-11 are 0, 2.5, 5, 10, 15, 20, 2 respectively. 5, 30, 50 or 100 mM (NH_Four)_TwoSO_FourIn a buffer containing Cleavase^{T M} Includes reaction products that result from incubating the substrate in the presence of the BN enzyme. lane 12 is 100 mM (NH_Four)_TwoSO_FourIncludes no enzyme control incubated in buffer containing No.   The results shown in FIG. 56 are for Cleavase.^TMThe reaction is (NH_Four)_TwoSO_FourThe presence of Is severely inhibited by 15 mM (NH_Four)_TwoSO_FourThe reaction is complete even with a small amount Totally inhibited, 5 mM (NH_Four)_TwoSO_FourOf the cleavage reaction at 20 mM KCl Degree is almost the same as 0 mM (NH_Four)_TwoSO_FourConsiderably lower than the degree of cleavage obtained in Was. However, 5 mM (NH_Four)_TwoSO_FourThe cleavage pattern obtained using_Four)_TwoSO_Fourof Incubation of 157 bp substrate in the absence or in KCl or NaCl Is the same as the cleavage pattern observed in Cleavase^TM The choice of salt involved in the reaction does not affect the nature of the site recognized by the enzyme. Suggest that E) Temporal change of double-strand cleavage reaction   To determine how fast the double-strand cleavage reaction is completed, a single substrate A fixed amount of Cleavase^TMInks at various times in the presence of BN enzyme Was added. A 157 bp fragment of exon 4 of the tyrosinase gene (SEQ ID NO: 4) 0: Prepared by the method of Example 22a) Approximately 100 fmoles were Place in a heart tube (BioRad, Richmond, CA) in a volume of 6.25 μL in sterile distilled water. Was. The tube was heated at 95 ° C for 15 seconds and quenched to 45 ° C as described in section B).   2.7X CFLP^TMBuffer, pH 7.5 (to give a final concentration of 1X), 0.53 mM MnCl_Two(Final concentration 0 .2mM), Cleavase^TMBN [1X dilution buffer (0.5% NP40,0 50 ng / μL in 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) The cleavage reaction was initiated by adding 3.75 μL of the enzyme mixture containing 0.5 μL. . Final reaction volume was 10 μL. Parallel with no enzyme (ie, uncleaved) control And stopped at 5 or 120 minutes, this was Cleavase )^TMThe difference was that sterile distilled water was used instead of BN.   The cleavage reaction was stopped by adding 8 μL of stop buffer at the following times: : 5 seconds, 1, 2, 5, 10, 15, 20, 30, 60 or 120 minutes. The sample Heat at 72 ° C. for 2 minutes and apply 4 μL of each reaction to a 10% polyacrylamide gel (1 9: 1 cross-linking) and 7X urea in buffer containing 0.5X TBE Therefore, they were separated.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 57 shows the obtained autoradiograph. Shown in   In FIG. 57, lane 1 contains an enzyme-free control incubated at 45 ° C. for 5 minutes. Shows the mobility of uncleaved template DNA. Lanes 2-10 are 5 seconds, 1, 2, 5, 10, 15, 20, 30, 60 (1 hour) or 120 minutes (2 hours) Cleavase^TMProduced by incubating substrate in the presence of BN enzyme Includes cleavage fragments. Lane 11 shows the enzyme after incubation at 45 ° C. for 120 minutes. Includes no control.   The results shown in FIG. 57 are for Cleavase.^TMMediated by the BN enzyme This indicates that cleavage of the double-stranded DNA template is rapid. Completely cleaved within one minute And is substantially complete. Example of cleavage of single-stranded DNA template (Example 1 Unlike 1c), a very slight cleavage is detected after 5 seconds. This reaction does not It contained 1/10 of the enzyme used in the reaction described in Example 11c. further, Incubation of single-strand cleavage reaction for a long time increases the number of short-cleaved fragments. (Example 20e), however, the sig- Null disappeared completely (FIG. 57, lane 10). The result is probably that the reassociated chains May be due to enzymatic cleavage of a 5 'biotin molecule from E. coli. This result is Regardless of whether the reaction is carried out for 1 minute or 30 minutes, the cleavage of double-stranded DNA It is therefore important to show that the same cleavage pattern occurs. Ie Even if the incubation time is 30 times, the complete cleavage product (ie C), and therefore a double-stranded CFLP^TMIncubation in reaction You don't need to control the exact time.   The results shown in FIG. 58 include the time course of cleavage reactions performed at various enzyme concentrations. H A double-stranded 157 bp fragment of exon 4 of the rosinase gene (SEQ ID NO: 27) 0 fmol was lysed in a 200 μL thin-walled microcentrifuge tube (BioRad, Richmond, CA). Bacteria were placed in a volume of 6.25 μL in distilled water. Tubes were placed in 9 as described in Example 22b. Heated at 5 ° C for 15 seconds and quenched to 45 ° C. 2.7X CFLP^TMBuffer, pH 7.5 (final concentration 1 X), 0.53 mM MnCl_Two(To give a final concentration of 0.2 mM), Cleavase )^TMBN [1X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50m 50, 100, 200 or 500 ng / μL in M KCl, 10 μg / ml BSA) Give 25, 50, 100 or 250 ng elementary] 3.75 μL of enzyme mixture containing 0.5 μL To initiate the cleavage reaction. Final reaction volume was 10 μL. Enzyme-free A control was prepared in parallel, which was Cleavase.^TMSterilization instead of BN The difference was that distilled water was used and stopped after 1 minute at 45 ° C.   After 5 seconds or 1 minute, the cleavage reaction was stopped by adding 8 μL of stop buffer. The sample was heated at 72 ° C. for 2 minutes and 4 μL of each reaction was added to 10% polyacrylamide. Use 7M urea in buffer containing Dogel (19: 1 cross-linking) and 0.5X TBE Separated by electrophoresis.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 58 shows the obtained autoradiograph. Shown in   In FIG. 58, the lane indicated by "M" contains the molecular weight marker described in Example 8a. . Lane 1 contains the no enzyme control. Lanes 2 and 3 each have 25 ng of Cleavase^TMReaction products generated by cleavage of substrate in the presence of BN enzyme. Contained product, reaction in lane 2 stopped after 5 seconds and in lane 3 after 1 minute . Lanes 4 and 5 are 50 ng each of Cleavase.^TMBN enzyme The reaction in lane 4 for 5 seconds, including the reaction product resulting from cleavage of the substrate in the presence of Later, in lane 5, it stopped after 1 minute. Lanes 6 and 7 are 10 0 ng of Cleavase^TMProduced by cleavage of substrate in the presence of BN enzyme The reaction in lane 6 stopped after 5 seconds and in lane 7 after 1 minute Stopped. Lanes 8 and 9 each contain 250 ng of Cleavase (Cleava se)^TMLane 8 contains the reaction products generated by cleavage of the substrate in the presence of the BN enzyme. Was stopped after 5 seconds, and in Lane 9 after 1 minute.   The results shown in FIG. 58 show that the double-stranded DNA cleavage rate increased with increasing enzyme concentration. To do so. As the enzyme concentration increased, the amount of uncleaved DNA remaining after one minute Note that it decreases accordingly. As shown below, in FIG. The enzyme concentration involved has no effect on the resulting cleavage pattern. 250 ng Reaction (FIG. 58, lanes 8 and 9) and the short digestion described in Example 11c. When compared, the amount of enzyme is very small, rather than the nature of the substrate, whether it is double-stranded or single-stranded. It suggests controlling the degree of initial cleavage. F) Temperature titration of double-strand cleavage reaction   To determine the effect of temperature changes on cleavage patterns, the tyrosinase gene 157 bp fragment of exon 4 of SEQ ID NO: 27 (SEQ ID NO: 27) Cleavase)^TMIncubated for 5 minutes at various temperatures in the presence of BN enzyme. Substrate DN A About 100 fmol (prepared as described in Example 22a) was added to 200 μL of a thin-walled micro Place in a centrifuge tube (BioRad, Richmond, CA) in a volume of 6.25 μL in sterile distilled water. Was. The tube was heated at 95 ° C. for 15 seconds and 37, 40, 45, 50, 55, 60, 65, Cooled to either 70, 75 or 80 ° C. 2.7X CFLP^TMBuffer solution, pH 7.5 (final Concentration 1X), 0.53mM MnCl_Two(Final concentration 0.2mM MnCl_Two), Cree Cleavase^TMBN [1X dilution buffer (0.5% NP40, 0.5% Tween20, 20 mM Tr 50 ng / μL in is-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) The cleavage reaction was started by adding 3.75 μL of the mixture. Enzyme mixture While keeping a small amount of the individual enzyme mixture at room temperature before adding it to the reaction. I got it. At the end of the experiment, a second reaction was performed at 37 ° C, which may have occurred during the experiment. No loss of enzyme activity was controlled. An enzyme-free control was prepared in parallel, Cleavase^TM37 ° C or 80 ° C using sterile distilled water instead of BN They differed in that they were incubated at different times. Add 8 μL of stop buffer To stop the reaction.   The sample was heated at 72 ° C. for 2 minutes and 5 μL of each reaction was added to 10% polyacrylic acid. Using midgel (19: 1 cross-linking) and 7M urea in buffer containing 0.5X TBE Separation by electrophoresis.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 59 shows the obtained autoradiograph. Shown in   In FIG. 59, the lane indicated by "M" contains the molecular weight marker described in Example 8a. . Lane 1 contains the enzyme-free control after 5 minutes incubation at 37 ° C. Leh Incubations 2 and 3 were incubated at 37 ° C., performed at the beginning and end of the experiment, respectively. Including reactions. Lanes 4-13 are 40, 45, 50, 55, 60, 65, 70, Includes reactions incubated at 75 or 80 ° C [2 samples at 80 ° C RU: Chill Out 14^TM(MJ Research, Watertown, MA) not covered Second, after adding the enzyme mixture, Chill Out 14^TM20 μL]. Lane 14 Contains an enzyme-free control incubated at 80 ° C. for 5 minutes.   FIG. 59 shows that double-stranded DNA substrate cleavage proceeds most efficiently at low temperatures. . In the range of 37 ° C to 60 ° C, the cleavage signal responds to the incubation temperature. And the pattern distribution changed smoothly. Some cleavage products can be used to Others were much more pronounced at low temperatures, while others appeared only at a low temperature. Many Minutes, Cleavase at one end of the temperature range^TMA substrate for the BN enzyme Some structures may not be suitable at the other end. Cleavage structure, as expected Due to the melting of the structure, the generation of cleavage fragments gradually decreased in the hottest region. 7 Above 0 ° C., cleavage products are limited to small fragments, which may be due to large denaturation of the substrate To do that. When using long DNA (350-1000 nucleotides) Was found to produce a useful cleavage pattern up to 75 ° C.   These results indicate that cleavage reactions can be performed over a fairly wide range of temperatures using double-stranded DNA substrates. Can be implemented. As in the case of the single-strand cleavage reaction, two It is important that the chains can be cleaved. Single strong secondary structure governs cleavage pattern Changes in bases do not result in instability and thus hinder the detection of mutations. Therefore The use of high temperatures can cause these rigid structures to be on the verge of instability, and The effect of various changes can be maximized to reveal differences in cleavage patterns. Ma This result can lead to block drift and other equipment parts heating in useful temperature ranges. Changes in the reaction temperature do not result in a large change in the cleavage pattern. It also shows that G) Cleavase in double-strand cleavage reaction^TMTitration of BN enzyme   Cleavase in double-strand cleavage reaction^TMEffect of concentration change of BN enzyme The fruit was tested. A 157 bp fragment of exon 4 of the tyrosinase gene (SEQ ID NO: 2) 7: Prepared as described in Example 22a) Approximately 100 fmol was added to 200 μL 2. Place in a centrifuge tube (BioRad, Richmond, CA) at a volume of 6.25 μL in sterile distilled water. Was. The tube was heated at 95 ° C for 15 seconds and quenched to 45 ° C.   2.7X CFLP^TMBuffer, pH 7.5 (to give a final concentration of 1X), 0.53 mM MnCl_Two(Final concentration 0.2 mM MnCl_Two), Cleavase^TMBN [1X dilution buffer (0.5% NP4 2,10% in 0, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) , 50, 100, 200 or 500 ng / μL, which means that the amount of enzyme added to the reaction is 1, 5, 10, Give 25, 50, 100 or 250 ng] 3.75 μl of enzyme mixture containing 0.5 μl Initiated the cleavage reaction. An enzyme-free control was prepared in parallel, Cleavase^TMThe difference is that a IX dilution buffer is used instead of BN. After 5 minutes at 45 ° C., the cleavage reaction was initiated by adding 8 μL of stop buffer. Stopped. The sample was heated at 72 ° C. for 2 minutes and 4 μL of each reaction was added to 10% 7M urine in a buffer containing acrylamide gel (19: 1 cross-linking) and 0.5X TBE Separation was carried out by electrophoresis using element.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 60 shows the obtained autoradiograph. Shown in   In FIG. 60, the lane indicated by "M" contains a molecular weight marker. Lane 1 shows no enzyme Includes controls and shows the mobility of uncleaved substrate. Lanes 2-8 are 1, 5, 10 , 25, 50, 100 or 250 ng of Cleavase^TMDue to reactions involving BN enzyme With the cleavage product coming.   These results show that even if the amount of enzyme used in the reaction differs by 50 times or more, 157 bp The same cleavage pattern can be obtained using tyrosinase DNA substrate (SEQ ID NO: 27) Indicates that Therefore, double-strand cleavage requires the control of reaction conditions in a laboratory. Not ideal to do in a clinical laboratory. However, observed with single-stranded substrates In contrast to (Example 11e), the double-stranded template was run at moderate enzyme concentrations. Note that the cleavage is optimal at this time. In double-stranded reactions, Ze (Cleavase)^TMThe gradual disappearance of the signal with increasing BN concentration is due to recombination This may be because the 5 'biotin label is cleaved from the end of the resulting double-stranded template.                                 Example 23             Determination of optimal pH for single- and double-strand cleavage reactions   Two types of primer-independent cleavage reactions (ie, single-stranded and double-stranded cleavage reactions) Cleavase at various pH ranges to determine the optimal pH conditions for (Cleavase)^TMA reaction buffer was prepared. A) Determination of optimal pH for single-strand cleavage reaction   Cleavase used to cleave single-stranded substrates^TMThe reaction (ie, C FLP^TM) The effect of changing the pH of the buffer was tested. 6M N in 1M MOPS solution, pH 6.3 By titration with aOH, the pH of 6.3, 7.2, 7.5, 7.8, 8.0 and 8.2 was 0.5M M Several 10X buffer solutions were prepared using OPS. Next, a 0.5M solution will be prepared for each pH The volume was adjusted as follows.   A single-stranded substrate prepared from the sense strand of exon 4 of the tyrosinase gene (SEQ ID NO: No. 34: Prepared as described in Example 8a) Approximately 100 fmol was 1X CFLP of various pH in microcentrifuge tubes (BioRad, Richmond, CA).^TMBuffer 15 μL, and 1.33 mM MnCl_Two(Giving a final concentration of 1 mM). Final reaction volume Was 20 μL. The reaction mixture was heated at 95 ° C for 5 seconds and quenched to 65 ° C. Again And 1X CFLP with appropriate pH^TMBuffer solution (MnCl_Two(Not including) Cleavase ase) T^MBN [1X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 ng / μL in 50 mM KCl, 10 μg / ml BSA)]. The reaction was started by the addition. Run 20 μL of salt-free, enzyme-free control in parallel And incubated at 65 ° C. at each indicated pH. This reaction is cleaved Cleavase^TMContains sterile distilled water instead of BN enzyme, all reaction components before denaturation Was different. After 5 minutes, add 16 μL of stop buffer To stop the reaction.   The sample was then heated at 72 ° C. for 2 minutes and 7 μL of each reaction product was described in Example 8a. 10% polyacrylamide gel (19: 1 cross-linking) and 0.5 × T Separation was achieved by electrophoresis using 7M urea in buffer containing BE.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8a. I put the membrane. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline 1X I-Block (Tropi) conjugated with phosphatase (United States Biochemical) x, Bedford, MA), washed and CD as described in Example 20a. P-Star^TM(Tropix, Bedford, MA) and exposed the film to X-ray film. Washing with distilled water was omitted. FIG. 61 shows the obtained result.   In FIG. 61, panels A and B include reactions using single-stranded DNA substrates. Panel A Shows five pairs of reactions. In each case, the first lane of the pair was an enzyme-free control. Yes, the second lane is a single-strand cleavage reaction. Lanes 1 and 2 are at pH 6.3; Lanes 3 and 4 have a pH of 7.2; lanes 5 and 6 have a pH of 7.8; 8.0; Lanes 9 and 10 show the reaction products obtained using the reaction buffer at pH 8.2. Indicates an object. Panel B shows pH 7.5 (lanes 1 and 2 show uncut and cut, respectively). And pH 7.8 (lanes 3 and 4 show uncleaved and cleaved, respectively) 3 includes the results of another experiment comparing cleavage reactions performed with the reaction buffers of FIG.   The results shown in FIG. 61, panels A and B, show that cleavage of the single-stranded DNA template was relatively small. Suggests that it is sensitive to pH changes. Optimal reaction from pH 7.5 to 8.0 There was a pH. MOPS pK_aIs 7.2, so this value to support cleavage The closest pH, pH 7.5, was determined to be optimal for single-strand cleavage reactions. B) Determination of optimum pH for double-strand cleavage reaction   Cleavase used for cleavage of double-stranded substrates^TMThe reaction (ie, C FLP^TM) The effect of changing the pH of the buffer was tested. As described in section A) Several 10X buffer solutions using 0.5M MOPS with pH 7.2, 7.5, 7.8 and 8.0 Was prepared. A double-stranded 157 bp fragment of exon 4 of the tyrosinase gene (sequence No. 27: Prepared as described in Example 8) Approximately 100 fmol was Tubes (BioRad, Richmond, CA) in a total volume of 6.25 μL. 9 tubes Heated at 5 ° C for 15 seconds and cooled to 45 ° C. 2.7X CFLP^TMBuffer, pH 7.5 (final concentration 1 X), 0.53 mM MnCl_Two(Final concentration 0.2mM MnCl_Two), Cleavase (Cle avase)^TMBN [1X dilution buffer (0.5% NP40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0 , 50 mM KCl, 50 μg / ml in 10 μg / ml BSA). To initiate the cleavage reaction.   Incubate the cleavage reaction for 5 minutes and reverse by adding 8 μL of stop buffer. Stopped responding.   The sample was then heated at 72 ° C. for 2 minutes and 4 μL of each reaction product was added to 10% polyacrylic acid. Luamide gel (19: 1 cross-linking) and 7M urea in buffer containing 0.5X TBE Separated by electrophoresis used.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8b. I put the membrane. Transfer the DNA to the membrane, dry the membrane and add 1X I-Block blocking buffer. Wash, wash and expose membrane to X-ray film as described in Example 20a. Although light was emitted, washing with distilled water was omitted. FIG. 62 shows the obtained autoradiograph. , Panels A and B.   In flannel A of FIG. 62, lanes 1 and 2 show the reactions performed in a buffer at pH 8.2. Contains cleavage products (lane 1 contains cleavage reaction, lane 2 is uncleaved control) . Lanes 3 and 4 contain the cleavage products of the reaction performed in pH 7.2 buffer. ー Lane 3 contains a cleavage reaction, lane 4 is the uncleaved control). In panel B, the lane 1 and 2 contain the cleavage products of the reaction performed in pH 7.5 buffer (lane 1 Uncut control, lane 2 contains cleavage reaction). Lanes 3 and 4 have a pH of 7.8. Containing the cleavage products of the reaction performed in the same buffer (lane 3 is the uncleaved control; Is the cleavage reaction).   The results shown in FIG. 62, panels A and B show that the cleavage of double-stranded DNA This suggests that it was not sensitive to changes in the pH range of the conditions. However, one Since strand DNA cleavage was sensitive to pH change, it was determined to be optimal for single-strand cleavage. The buffer conditions used were used in the following double-strand cleavage experiments.                                 Example 24               The presence of competing DNA does not alter the cleavage pattern   Effect of the presence of competitor (ie, unlabeled substrate) DNA on the cleavage reaction Was tested. 157 nucleotide fragment derived from the sense strand of human tyrosinase gene A cleavage reaction was performed using (SEQ ID NO: 34) and human genomic DNA. It is shown below The results show that the presence of non-substrate DNA indicates that CFLP^TMAnything in the pattern Indicates no effect. A) Preparation of substrate DNA and cleavage reaction   157 nucleotide single-stranded wild-type tyrosiner containing a biotin label at the 5 'end Ze substrate (SEQ ID NO: 34) was prepared as described in Example 9. Tris-HCl, pH 8.0; human genomic DNA (Promega) present at 235 μg / ml in 1 mM EDTA And resuspended in Tris-HCl, pH 8.0; 1 mM EDTA at a final concentration of 400 μg / ml. This DNA is converted to standard CFLP^TMAs a competitor in the single-stranded reaction (described in Example 9) Used. Tyrosinase DNA substrate (SEQ ID NO: 34) and human genomic DNA_Two Mix in O in a final volume of 6 μL. Heat the sample at 95 ° C for 10 seconds to DNA Is denatured, cooled to a target temperature of 65 ° C., and 5X CFLP^TMBuffer solution, pH 7.5, 2 μL, 10 mM MnCl_Two1 μL and enzyme Cleavase in dilution buffer^TM1μ of BN L (25 ng) was added. After 5 minutes at 65 ° C., add 6 μL of stop buffer to The reaction was stopped and 5 μL of each sample was separated on a 10% denaturing polyacrylamide gel. Was. Transferred to membrane and DNA visualized as described in Example 19. B) The presence of genomic DNA is CFLP^TMDo not change the pattern   FIG. 63 shows 0 μg / ml (lane 2), 20 μg / ml (lane 3), 40 μg / ml (lane 4), 80 μg / ml (lane 5), 120 μg / ml (lane 6) and 200 μg / ml (lane 7) ) In the presence of unlabeled human genomic DNA, the sense strand of the wild-type tyrosinase substrate ( 34 shows the pattern obtained corresponding to the cleavage product of SEQ ID NO: 34). Lane 1 is yeast Cleavase^TMUncut control in the absence of BN, indicated by "M" The lane contains the molecular weight markers prepared as described in Example 8.   FIG. 63 shows that the presence of genomic DNA during the cleavage reaction results in the location of product bands or Indicates that none of the relative intensities was changed. However, during the reaction, As the amount of foreign DNA increases, the efficiency of the cleavage reaction decreases and the overall pattern Strength decreased. These results are based on Cleavase^TMBN enzyme is not special Binds to foreign DNA, which has the effect of reducing the effective enzyme concentration during the reaction Can be explained. This effect was observed when the genomic DNA concentration in the reaction was 120 μg / ml or less. It became remarkable when it turned up [FIG. 63, 120 μg / ml (lane 6) and 200 μg / ml (Lane 7)]. Under these conditions, genomic DNA is compared to specific substrate DNA In excess of 20000-fold, but under these conditions CFLP^TMPa The turn was still recognizable. CFLP in the presence of genomic DNA^TMThe pattern is stable What has been observed is that non-specific DNA can significantly alter the structure of substrate DNA Or substrate and Cleavase^TMChange interaction with BN enzyme It excludes the possibility of doing so.                                 Example 25               CFLP^TMReaction can be performed using various enzymes   In the above examples, 5 'nuclease derived from Taq DNA polymerase was used. Cleavase^TMSet of cleavage fragments characteristic of BN enzymes from nucleic acid substrates The ability to produce In the following experiments, we used a number of other enzymes Figure 4 shows that a set of cleavage products characteristic of a particular nucleic acid can be generated. A) Cleavage fragments generated by other DNA polymerases from the genus Thermus turn   5 'Nucleic acid associated with DNAP other than Taq DNA polymerase (DNAP) Determining whether the enzyme activity can generate a unique cleavage pattern from a nucleic acid substrate To this end, DNAPs from two species of the genus Thermus were tested. Thermath Flavas (Thermus flavus) ["Tfl", Kaledin et al. Biokhimiya 46: 1576, 1981:  Obtained from Promega Corp., Madison, WI]. Ruth (Thermus thermophilus) ["Tth", Carballeira et al., Biotechniq ues 9: 276, 1990: Myers et al., Biochem. 30: 7661, 1991: U.S. Biochemical From Cleaveland, OH] using the appropriate cleavage pattern (i.e., The ability to generate a pattern that can be used to characterize a particular nucleic acid substrate The force was tested.   The ability of these other enzymes to cleave nucleic acids in a structure-specific manner is determined by the D Under conditions reported to be optimal for NA synthesis, the exogene of the tyrosinase gene Using a single-stranded 157 nucleotide fragment (SEQ ID NO: 34) derived from the sense strand of Tested.   A 157 nucleotide fragment from the sense strand of exon 4 of the tyrosinase gene ( SEQ ID NO: 47: Prepared as described in Example 10a) 1X CFLP in a thin-walled microcentrifuge tube (BioRad, Richmond, CA)^TMBuffer, pH 8.2 and 1.33 mM MnCl_Two(Giving a final concentration of 1 mM) and giving a final concentration of 0 or 50 mM KCl was added. Final reaction volume was 20 μL. Apply sample at 95 ° C for 5 seconds Heated and cooled to 65 ° C. Prepare 20 μL of salt-free, enzyme-free control in parallel, This is Cleavase^TMContains sterile distilled water instead of BN enzyme, all The reaction components differed in that they were added before denaturation at 95 ° C.   1X CFLP^TMThe indicated enzyme mixture in buffer, pH 8.2 (see below) The reaction is opened by adding 5 μL of the diluted enzyme mixture containing 25 or 5 units. Started. After 5 minutes, the reaction was stopped by adding 16 μL of stop buffer.   Heat the sample at 72 ° C. for 2 minutes and 7 μL (for a sample digested with Tfl) Alternatively, 5 μL (for a sample digested with Tth) was applied as described in Example 8a. Contains 10% polyacrylamide gel (19: 1 cross-linking) and 0.5X TBE Separation was achieved by electrophoresis using 7M urea in buffer.   After electrophoresis, the gel plate was separated and treated with nylon as described in Example 8a. I put the membrane. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline 1 conjugated with phosphatase (United States Biochemical, Cleaveland, OH) Blocked in XI-Block (Tropix, Bedford, MA), washed and described in Example 20a. CDP-Star as shown^TM(Tropix, Bedford, MA) The film was exposed to light, but washing with distilled water was omitted. The obtained results are shown in FIGS. It is shown in FIG.   In FIG. 64, lane 1 contains an enzyme-free control and shows the mobility of uncleaved DNA. Les 2-5 show the cleavage products from the reaction incubated with Tfl DNAP. Including. The reactions shown in lanes 2 and 3 each contained a Tfl DNAP5 unit. , The sample in lane 2 was incubated in a reaction buffer containing 0 mM KCl, The sample in lane 3 was incubated in a reaction buffer containing 50 mM KCl. Leh The reactions shown in steps 4 and 5 each contain 1.25 units of Tfl DNAP, Sample was incubated in reaction buffer containing 0 mM KCl. The 5 sample was incubated in a reaction buffer containing 50 mM KCl.   In FIG. 65, lanes 1 and 2 are incubated with 1.25 units of Tth DNAP. Includes cleavage products from the reaction. The sample in lane 1 contains 0 mM KCl. The sample in lane 2 was incubated in 50 mM KCl Incubated in buffer. Lanes 3 and 4 are Tth DNAP5 units Includes cleavage products from reactions incubated with. Lane 3 sample is 0 mM Incubation in a reaction buffer containing KCl, the sample in lane 4 was 50 mM KC Incubated in reaction buffer containing l.   64 and 65 show that both Tth DNAP and Tfl DNAP are clear. Cleavase^TMStructure-specific endos with properties similar to those seen with the BN enzyme It has nuclease activity. Comparing the results shown in FIGS. 64 and 65, Tth DNAP produces a more efficient cleavage pattern under the reaction conditions tested Indicates that Cleavase (Cleavase) was used to determine the cleavage pattern generated by Tth DNAP. se)^TMCompared to the cleavage pattern generated by the BN enzyme, these two enzymes are substantially This suggests that it recognizes the same structure [Figure 66, lane 2 (cleavease (Cl eavase)^TMCompare BN with Figure 65 (Tth DNAP)]. B) Enzymes characterized as 3 'nucleases produce unique cleavage patterns Can be used for   Enzymes with 3 'nuclease activity can also generate unique cleavage patterns To determine whether or not DNAP (with 5 'nuclease) The cleavage reaction was tested with the enzyme. Exonuclea from Escherichia coli Escherichia coli III (E. coli Exo III) is ligated with the sense chain of exon 4 of the tyrosinase gene. The 157 nucleotide fragment (SEQ ID NO: 34) was tested in a cleavage reaction. ratio As a comparison, this substrate (SEQ ID NO: 34) and Cleavase^TMBN enzyme Reactions were also prepared.   A 157 nucleotide fragment from the sense strand of exon 4 of the tyrosinase gene ( SEQ ID NO: 34: prepared as described in Example 8a) 1X CFLP in thin-walled microcentrifuge tubes (BioRad, Richmond, CA)^TMBuffer solution, pH 8.2 and 1.33 mM MnCl_Two(Giving a final concentration of 1 mM) and K giving a final concentration of 0 or 50 mM Cl was added in a volume of 15 μL. Final reaction volume was 20 μL.   The sample was heated at 95 ° C for 5 seconds and quenched to 37 ° C. Salt-free, enzyme-free control Prepare 20 μL in parallel, which contains Cleavase^TMBN enzyme Instead, it contains sterile distilled water, with the difference that all reaction components are added before denaturation at 95 ° C. Had become.   100 f in a buffer containing 0 mM KCl (SEQ ID NO: 34) Mole and Cleavase^TMPrepare a reaction tube containing 50 ng of BN enzyme, and No. 21 (ie, incubation at 95 ° C. for 5 seconds to change And then cooled to 65 ° C, added enzyme and incubated at 65 ° C for 5 minutes).   1X CFLP^TMBuffer, pH 8.2 (MnCl_TwoExo III (United States)  Biochemical, Cleveland, OH) Dilute enzyme mix containing 1.25 or 200 units The reaction is started by adding 5 μL of the mixture to 15 μL of the reaction, and the reaction is allowed to flow for 5 minutes. Incubated. After 5 minutes at 37 ° C., add 16 μL of stop buffer. To stop the reaction.   The sample was heated at 72 ° C. for 2 minutes and 5 μL was added to 1 μl as described in Example 8a. Buffer containing 0% polyacrylamide gel (19: 1 cross-linking) and 0.5X TBE Separated by electrophoresis using 7M urea in.   After electrophoresis, the gel plate was separated and analyzed on a Nylon gel as described in Example 10a. The film was put on. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline Phosphatase (United States Biochemical, Cleaveland, OH) Blocked in 1X I-Block (Tropix, Bedford, Mass.), Washed, Example 20a CDP-Star as described in^TM(Tropix, Bedford, MA) The film was exposed, but the washing with distilled water was omitted. FIG. 66 shows the obtained result. Show.   Lane 1 of FIG. 66 contains an enzyme-free control and shows the mobility of uncleaved DNA. Leh 2 is Cleavase^TMIncubating the substrate with the BN enzyme Comparison of patterns generated by two different enzymes, including cleavage fragments generated by provide. Lanes 3-6 are produced by incubating the substrate with Exo III. Including cleaved fragments. Lanes 3 and 4 each contain 200 Exo III units. The reaction in lane 3 contains the reaction product resulting from the The reaction in Lane 4 was performed in a buffer containing 50 mM KCl. Leh 5 and 6 are the reaction products generated in the reaction containing 1.25 units of Exo III, respectively. The reaction in lane 5 was performed in a buffer containing 0 mM KCl, The reaction was performed in a buffer containing 50 mM KCl.   The results shown in FIG. 66 indicate that Exo III was isolated when incubated with single-stranded DNA substrate. It shows that it produces a special cleavage pattern. Exo III pattern is cleaved Cleavase^TMIt was completely different from the pattern produced by the BN enzyme. As shown in FIG. The results show that significant differences in the cleavage pattern that occur with Exo III are included in the reaction. It is also shown that it is observed depending on both the enzyme and KCl concentration. C) Ability of alternative enzymes to identify single base changes   In sections A) and B) above, Cleavase^TMEnzymes other than BN enzyme Can produce unique patterns when incubated in the presence of nucleic acid substrates. Showed that you can. Tth DNAP and E. coli. coli Exo III is single-stranded D Exists in DNA substrates of the same size as it produces a unique cleavage pattern at NA The ability of these enzymes to detect single base changes was tested. Same as Example 9 Similarly, in exon 4 of the human tyrosinase gene, multiple single point mutations (point Mutation) has been identified, so select this gene as a model system did.   Three single-stranded substrate DNAs were prepared: all three of these substrates were bi-terminal at the 5 'end. It contained an otine label. Wild-type substrate is derived from the sense strand of the human tyrosinase gene 157 nucleotides (SEQ ID NO: 34). Using two mutation-containing substrates Was. Both the 419 substrate (SEQ ID NO: 41) and the 422 substrate (SEQ ID NO: 42) This is described in Example 9. Single-stranded DNA having a biotin label at the 5 'end is Each substrate was prepared using the asymmetric PCR described in Example 8a, except that two Single stranded PCR product, but not strand product, was recovered from the gel.   The cleavage reaction was performed as follows. Each substrate DNA (about 100 fmol) In a 00 μL thin-walled microcentrifuge tube (BioRad, Richmond, CA), 10 mM MOPS, pH 8.2. And 1.33 mM MnCl_TwoPlaced at 5 μL (giving a final concentration of 1 mM). Enzyme-free controls Prepare in parallel with the wild-type DNA fragment and incubate at 65 ° C for the indicated times. And this includes Cleavase^TMSterile distilled water instead of BN enzyme And all reaction components differed in that they were added before denaturation at 95 ° C. reaction The tube was heated at 95 ° C. for 5 seconds to denature the substrate, and then for reactions containing Tth DNAP The reaction was rapidly cooled to 65 ° C. and 37 ° C. for the reaction containing Exo III.   5 μL of MnCl_TwoTth DNAP or Exo in 10 mM MOPS, pH 8.2 By adding a diluted enzyme mixture containing 1.25 units of any of the enzymes of III. To initiate the cleavage reaction. Allow the enzyme solution to come to room temperature before adding to the cleavage reaction . After 5 minutes at 65 ° C., the reaction was stopped by adding 8 μL of stop buffer. The sample was heated at 72 ° C. for 2 minutes and 7 μL of each reaction was added to 10% polyacrylamide gel. Using 7 M urea in buffer containing 0.5X TBE (19: 1 cross-link) and 0.5X TBE Separated by electrophoresis.   After electrophoresis, the gel plate was separated and analyzed on a Nylon gel as described in Example 10a. The film was put on. Transfer the DNA to the membrane, dry the membrane, and use streptavidin-alkaline 1-X I-Block (Tro) conjugated with a reactive phosphatase (United States Biochemical) pix, Bedford, MA), washed, and as described in Example 20a. CDP-Star^TM(Tropix, Bedford, MA) and exposed the film to X-ray film However, washing with distilled water was omitted. FIG. 67 shows the obtained result.   In FIG. 67, lanes 1-3 are wild-type and mutant mutants of the tyrosinase gene, respectively. 419 and the mutant 422 allele are inked with Tth DNAP. Includes cleaved fragments generated by hybridization. Lanes 4-6 are wild type and mutant, respectively. 419 and mutant 422 substrate were supplemented with Exo III and 0 mM KCl. Cleaved fragments generated by incubation in a buffer solution containing Lanes 7-9 are Any of the wild-type, mutant 419 and mutant 422 substrates were   Cleavage fragments generated by incubation in a buffer containing III and 50 mM KCl including. Lane 10 shows that wild-type DNA substrate was cleaved from Cleavase.^TMBN enzyme And a cleavage fragment produced by incubation in a buffer containing 0 mM KCl. This reaction involves three different enzymes (ie, Cleavase).^TMBN enzyme , Tth DNAP and Exo III). You. Lane 11 is the incubation with wild-type DNA substrate in the presence of 50 mM KCl. Includes control without enzyme.   The results in FIG. 67 indicate that both Tth DNAP and Exo III showed single-stranded D Shows that single base changes can be detected for wild-type DNA substrates in NA substrates . The pattern generated by Tth DNAP shows cleavedase for all three substrates. (Cleavase)^TMIt was comparable to the pattern produced by the BN enzyme (cleave Cleavase^TMSee FIG. 29 for a comparison with the patterns possible with the BN enzyme. When).   The pattern generated by Exo III is an enzyme derived from the genus Thermus (ie, Cleave Alease (Cleavase)^TMWhat is the pattern generated by BN enzyme and Tth DNAP) Was different. Furthermore, the pattern generated by cleavage of the DNA substrate by Exo III Was clearly different depending on the KCl concentration used in the reaction (see FIG. 67). Izu At these KCl concentrations, a unique pattern change was apparent for the 419 mutant. there were. As shown in FIG. 67, 40 mM in the 419 mutant at 0 mM KCl. A band appeared within the nucleotide region (lane 5); 419 at 50 mM KCl. The mutant contains an additional band within the 70 nucleotide region (lane 8). 42 2 In the mutant, no pattern change was recognized by Exo III digestion (compared to wild type). ); The difference in the ability of the E. coli Exo III enzyme to detect this single base change Relative position of the change with respect to the secondary structure, which acts as a substrate for the specific cleavage reaction, And the position of the label (5 'or 3') compared to the preferred cleavage site (5 'or 3') is connected with. FIG. D) Use of Drosophila RrpI enzyme to generate cleavage patterns it can   Another member of the Exo III family of DNA repair endonucleases An RrpI (N) from Drosophila melanogaster ugent, M .; Huang, S.-M. and Sander, M.S. Biochemistry 1993: 32, pp. 11445-114 52) was tested for its ability to generate a unique cleavage pattern on a single-stranded DNA template. The nature of this enzyme in the cleavage assay is not yet known, so various buffers Tested under conditions. Various amounts of enzyme (1 ng or 30 ng) were added to the tyrosinase gene About 100 fmol of the 157 nucleotide fragment of the sense strand of Song 4 (SEQ ID NO: 47) , 1mM MnCl_TwoOr 5mM MgCl_TwoIn and 1X CFLPTM buffer, pH 8.2 or 1X CFLP^TM Incubate in buffer, pH 7.8 and 10 mM NaCl. Heat the sample to 95 ° C , 1X CFLP^TAdding a diluted enzyme mixture containing 1 or 30 ng of RrpI in M buffer To start the reaction. The reaction was carried out at 30 ° C. for 5 or 30 minutes. The obtained knot The results (data not shown) indicate that this enzyme has a weak but unique cleavage pattern on the single-stranded DNA template. In the process. E) Rad1 / Rad10 complex can be used to generate cleavage patterns   S. Rad1-Rad10 endonuclease from cerevisiae (Rad1 / 10) is a specific 3 'peptide involved in nucleotide excision repair in yeast. This is a nuclease. This enzyme has two proteins, Rad1 and Rad 10 heterodimer. Rad1 and Rad10 alone are enzymes It has no sex. Rad1 / 10 recognized a structure containing a bifurcated DNA duplex. And cleaves the single-stranded 3 'arm at the end of the double strand (Bardwell, A.J. et. al., Science 265: 2082, 1994). In this sense, Rad1 / 10 is cleaved Cleavase^TMShares the same substrate specificity that BM enzymes do. However Rad1 / 10 cleaves the 3 'single-stranded arm in the double strand, whereas Cleavase^TMSince the BM enzyme cleaves the 5 'single-stranded arm, Rad 1/10 and Cleavase^TMDifferent cleavage products from BN enzyme You.   FIG. 68 shows a bifurcated DNA duplex (formed from the hairpin structure shown in the figure). FIG. 2 is a schematic diagram showing cleavage sites by these two enzymes. Fig. 68 The hairpin structure shown above has a 5 'nuclease (e.g., Cleavase (Cleaase) vase)^TMBN enzyme). The hairpin structure shown at the bottom of FIG. Indicates the site of cleavage by an enzyme (eg, Rad1 / 10) that cleaves at the single-stranded arm . Enzyme that cleaves at the 5 'single-stranded arm is cleaved^TMWith the 5 'enzyme The enzyme that cleaves at the 3 'single-stranded arm is called Cleavase.^TM3 'yeast Call it elementary.   Whether Rad1 / 10 protein can detect single base changes in DNA substrates Rad1 / 10 and Cleavase to determine^TMBN yeast The cleavage patterns resulting from cleavage of the DNA substrate by the element were compared. In this comparison Used the following substrates. Including a biotin label at the 5 'end as described in Example 9. A wild-type (SEQ ID NO: 3) derived from the sense strand of exon 4 of the human tyrosinase gene. 4) 419 mutant (SEQ ID NO: 41) and 422 mutant (SEQ ID NO: 42) ) A 157 nucleotide fragment obtained from the allele was generated.   Rad1 and Rad10 proteins are available from Errol C.I. Dr. Friedberg (The Univesit y of Texas Southwestern Medical Center, Dallas). Rad1 The / 10 complex contains Rad1 and Rad10 proteins in 1 × dilution buffer (0.5% N P40, 0.5% Tween 20, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 μg / ml BSA) Each protein was prepared by mixing so that the final concentration was 0.1 mM.   The cleavage reaction using Rad1 / 10 endonuclease was performed as follows. did. 15 ng of substrate DNA and Rad1 / 10 complex in 1 μl of 1 × dilution buffer (0.1 pmol) on ice with 10 μL of 10 mM MOPS, pH 7.8, 1 mM MnCl_TwoAnd mixed. The reaction was then incubated at 37 ° C. for 5 minutes. Add 6 μL of stop buffer to prevent cleavage. Stopped responding.   Cleavase^TMCleavage reaction using BN enzyme is Rad1 / 10 cleavage Performed exactly as described above, but with Cleavase^TMBN enzyme is 1 0 ng was added, and incubation at 37 ° C. was performed for 3 minutes. For each substrate DNA Uncut or enzyme-free controls were performed and prepared as described for reactions containing enzymes. However, sterile distilled water was added in place of the enzyme (data not shown).   Cleavage products (3 μL each) of 10% modified polyacrylic as described in Example 19 Separated by amide gel electrophoresis, transferred to a membrane, and visualized. Autoradius obtained The ograph is shown in FIG.   FIG. 69 shows wild-type tyrosinase substrate (SEQ ID NO: 34) (lanes 1 and 4), 4 19 mutant (SEQ ID NO: 41) (lanes 2 and 5) and 422 mutant (SEQ ID NO: 42) A production pattern corresponding to the cleavage product of the sense strand of (lanes 3 and 6). Indicates a turn. Lanes 1-3 show that three substrate DNAs were cleaved with Cleavase.^TM The cleavage pattern resulting from incubation with the BN enzyme and lanes 4-6 are 3 Pattern generated by incubating two substrate DNAs with Rad1 / 10 enzyme Is shown. The lane marked "M" is the molecular weight map prepared as described in Example 8. Including the   The results shown in FIG. 69 indicate that Rad1 / 10 enzyme was converted from substrate DNA (lanes 4-6). Shows that a unique cleavage pattern can be generated, and that cleavage of the substrate The average length of the resulting product is Cleavase^TMRaw by BN enzyme Indicates that it is longer than the first one. Importantly, the results shown in FIG. A single base substitution found in the body tyrosinase substrate results in otherwise similar cleavage. Demonstrates specific changes in tyrosinase substrates with cleavage patterns (Compare lanes 5 and 6 with lane 4). Rad 1/10 Digestion of the mutant 419 substrate by about 40 nucleotides or less compared to the wild type. The lower band is weaker and there is no band. On the other hand, mutant 4 Upon digestion of the 22 substrate, several new bands in the region of 42-80 nucleotides Has appeared. Since both enzymes used the same reaction conditions, these results indicate that R ad1 / 10 is Cleavase^TMDN recognized by BN enzyme A shows that the same differences in the secondary structure can be detected. Rad1 / 10 is chestnut Cleavase^TMA putter different from the cleavage pattern produced by the BN enzyme Which is cleaved at the 3 'end of the DNA hairpin, leaving the substrate at the 5' end. This is because a longer fragment is generated when an end marker is included.                                   Example 26                       Double-stranded DNA substrate was used               Detection of mutations in the human β-globin gene   The results shown in Example 13 were^TMFor cleavage of single-stranded DNA substrate by BN enzyme Indicates that a single base change in a fragment of the β-globin gene can be detected. You. In this example, the mutation in the β-globin gene was^TMB It shows that detection is possible by cleavage of the double-stranded DNA substrate using the N enzyme.   Using PCR, wild-type β-globin gene (SEQ ID NO: 56), mutant 1 (SEQ ID NO: 56) 58) and the 536 bp fragment obtained from mutant 2 (SEQ ID NO: 59), A double-stranded substrate DNA containing a radiolabel was generated. PCR amplification of these substrates is described in Example 13a. Were performed as described in Gel purification and isolation of the double-stranded fragment is described in Example 19a. Performed as described.   The cleavage reaction was performed as described in Example 19c. Briefly, 2 μl in TE Of stock DNA (80 ng) in 3 μl of H_TwoO and denatured at 95 ° C for 20 seconds. Modified D Cool NA to 70 ° C. and add 2 μl 5X CFLP^TMBuffer pH 7.5, 2 μl of 2 mM MnCl_TwoAnd 1 Enzyme Cleavase in μl (25ng) dilution buffer^TMAdd BN and start the cleavage reaction Was. After 1 minute, the cleavage reaction was stopped by adding 6 μl of stop buffer. 1 μl of Cle avase^TM1 μl H instead of BN enzyme_TwoUncut control as above except for using O A cleavage reaction was performed. The cleavage products (5 μl each) were run on a 6% denaturing polyacrylamide gel. Separated by electrophoresis, transferred to a membrane and visualized as described in Example 19. Profit The resulting autoradiograph is shown in FIG.   FIG. 70 shows wild-type β-globin 536 bp fragment (lane 4), mutant 1 fragment (lane 5) and The cleavage pattern corresponding to cleavage of the sense strand of the mutant 2 fragment (lane 6) is shown. Leh Nos. 1 to 3 show uncleaved controls of the wild type, mutant 1 and mutant 2 substrates, respectively. Lanes marked with “M” are biotinylated components produced as described in Example 8. Includes child mass markers.   As shown in FIG. 70, when compared to the wild-type cleavage pattern, Base substitution reduces the intensity of the band migrating near the uncleaved DNA (lane 5) I do. When compared to the wild-type cleavage pattern, the base substitutions present in mutant 2 Band in the region immediately above the major product band (approximately 174 nucleotides) disappears I do.   Regarding the above double-strand cleavage reaction, the single-chain β-globin DNA described in Example 13 Reaction conditions different from those used for cleavage of the substrate were used. For cleavage of double-stranded substrates The conditions used were lower MnCl compared to the conditions used in Example 13._TwoDark Degrees were used, no KCl was added, higher temperatures and shorter reaction times were used. two Cleavage patterns generated by cleavage of single-stranded and single-chain β-globin DNA differ slightly. However, the position of the pattern change for mutants 1 and 2 was shown in Example 13. The base substitution was detected in the case of both double strands. these Results show that DNA doubles caused by a single base substitution in a larger DNA substrate Subtle changes in secondary structure were made using both single-stranded and double-stranded forms of the DNA substrate. Cleavase^TMThis shows that it can be detected by the BN enzyme.                                   Example 27         Unknown by comparison with the library where the pattern exists       Identification of mutations in the human β-globin gene CFLP ^™ pattern   The results shown in Example 13 were^TMThe β-globin substrate tested by the BN enzyme This shows that each produces a unique pattern of cleavage products. Van Differences in wild-type pattern were seen between wild-type and each mutant, and different bands Mutation pattern is observed for each mutant, allowing the mutant to be distinguished from wild type As well as distinguish each variant from the others. Identification and Cleavase for classification^TMCompare the products of the reaction with the previously characterized variants To characterize the second set of β-globin variants, CFLP by comparison with the set analyzed in Example 13.^TMNumber using pattern If the variants in the second set are the same as in any of the first set, or Judged whether the set was unique. These isolates All have been described previously (these isolates are described in this example). (Specifically stated at the end.) Experiments.   A first set of five β-globin variants, the wild-type β-globin gene (SEQ ID NO: No. 56), or Mutant 1 (SEQ ID NO: 58), Mutant 2 (SEQ ID NO: 59) or Mutant 3 (SEQ ID NO: 59) CFLP from column number 57)^TMCompared with the pattern. Including these five new isolates The resulting plasmid was propagated and purified and the five β- Single-stranded substrate DNA 534 and 536 nucleotides long for each of the globin genes Was prepared. Perform a cleavage reaction and separate the reaction products as described in Example 13. did. The obtained autoradiograph is shown in FIG.   FIG. 71 shows two panels. Panel A is described in Example 13 (and 41 shows the reaction products from the β-globin isolate (shown in FIG. 40). Panel B is number 4, The reaction products of five additional isolates, labeled 5, 6, 7 and 8 are shown. Add "M" The marked lane is a biotinylated molecular weight marker produced as described in Example 8. including.   CFLP shown in Panel A^TMThe isolates shown in Panel B by comparison with the pattern Can be transformed. The banding pattern of isolate 4 (panel B, lane 1) is shown in panel A Same as that seen for the wild-type β-globin substrate shown in (lane 1). In contrast, isolate 8 (panel B, lane 5) shows the previously characterized variant 3 (panel A, lane 5). Lane 4) is equivalent and isolate 6 (panel B, lane 3) shows two of the patterns Regions 2 and 3 (panel A, lane 3) and 3 (panel A, lane 4) appears to have the characteristics of both isolates 5 and 7 (which Panel B, lanes 2 and 4), respectively, seem identical and are seen in panel A. It turns out that it shows no pattern.   Then, to confirm the relationship between the different isolates, the PCR primer [5'-bioti KM29 primer (SEQ ID NO: 54) and 5′-biotinylated RS42 primer (SEQ ID NO: 54) 55)] using fmole^TMDNA Sequencing System (Promega Corp., Madison, W The identity of the mutation in I) by primer extension sequencing according to the manufacturer's method Was identified. The sequencing reaction was performed using the β-globin CFLP described in Example 13b.^TMUsed for reaction Visualization was performed by the same procedure as used.   CFLP^TMTwo isolates that matched members of the original set by pattern analysis Also matched in the sequence. Isolate 4 has the same wild-type sequence (SEQ ID NO: 56). Isolate 8 is a duplicate of mutant 3 (SEQ ID NO: 57).   CFLP^TMAccording to the pattern, isolate 6 shows both variants 2 and 3 of the original set. It seems to have a similar change to the one. The sequence of mutant 6 (SEQ ID NO: 69) Mutant 3 refers to a single base change, silent C to T substitution at codon 3. It shows that it has. Variant 6 was found in Variant 2 (SEQ ID NO: 59) It also has a G to A substitution at codon 26, only four bases downstream. This mutation Is a secretory site that produces a fraction of mRNA encoding non-functional proteins. It has been shown to promote the price site [Orkin. S.H. et al. (1982) Nature, 300: 768]. Both mutant 6 and mutant 2 show a band migrating to about 200 nucleotides. (Eg, in mutant 2 the band disappears or becomes weaker , Mutant 6 separates into three weak bands), but these changes are identical. It is worth noting that it has no appearance. Suddenly four nucleotides away These CFLPs caused by mutations^TMChanges can be distinguished from each other.   The last two isolates 5 and 7 have the same sequence (SEQ ID NO: 70) and A single base change within the first intron at position 110 was revealed. This bump Mutations result in abnormal splices that result in premature termination of translation of the β-globin protein. [R.A. Spritz et al. (1981) Proc. Natl. Acad. Sci. USA. 78 : 2455]. CFLP for these variants^TMBand that disappears at Variant 1 (about 400 nucleotides) and And variant 2 (about 200 nucleotides) CFLP^TMBetween the indicating bands in the pattern, The actual mutations (nucleotide 334 from the 5 'end of the label) are nucleotides 380 and 380, respectively. It is worth noting that between the variants 1 and 2 at 207 and 207. like this , CFLP^TMThe analysis not only indicates the presence of a change, but also provides location information.   From the results shown in FIG. 71, the first four (wild-type and three mutant) β -Cleavase from each of the globin substrates^TMOf the cleavage products produced by the BN enzyme Another β-globin isolate was characterized using the unique pattern as a reference . Banding patterns show overall "tribal" similarity, with each specific mutation There are slight differences related to species (eg band disappearance or migration). Band formation Turn differences are seen between wild-type and each mutant, and different banding patterns N Is found in each variant, which not only allows the variant to be distinguished from the wild type, It also allows each variant to be distinguished from each other.                                   Example 28           Influence of order of addition of reactants on double-strand cleavage pattern   The cleavage reaction using a double-stranded DNA substrate is considered a two-step process. The first stage is Denaturation of the DNA substrate, the second step is the initiation of the cleavage reaction at the target temperature. Get The cleavage pattern that results is determined by the conditions during denaturation (eg, when DNA is denatured in water). Or denaturation in a buffer, etc.) and the conditions for initiating the reaction (eg, cleavage The reaction is initiated by the addition of the enzyme or MnCl_TwoIs started by the addition of The following experiment was performed because there is a possibility that it may change.   To investigate the effect of the addition of reactants on the resulting cleavage pattern, The reaction components (ie, DNA, CFLP^TMBuffer, MnCl_TwoAnd Cleavase^TMBN Enzyme) All combinations of mixing were varied. 536 bp fragment from wild-type β-globin gene A single DNA substrate containing a strip (SEQ ID NO: 56) was used. The substrate DNA is Manufactured as described in Example 26, containing a biotin label at the 5 'end.   8 different combinations used in the denaturation and start-up stages for the addition of reaction components The substrate was cleaved by different cleavage reactions. These reactions are described below.   FIG. 72 shows the cleavage of the sense strand of the wild-type β-globin 536-bp substrate (SEQ ID NO: 56). The resulting pattern generated is shown. In lane 1, substrate DNA (5 μl H_TwoDenature 40 fmole of DNA in 1 μl of TE mixed with O) at 95 ° C for 10 seconds and cool to 55 ° C. 2 μl 5X CFLP containing 150 mM KCl^TMBuffer, 1 μl 2 mM MnCl_TwoAnd 1 μl ( 50ng) Cleavase^TMThe reaction was started by adding the BN enzyme. In lane 2 The DNA with 2 μl of 5X CFLP^TMDenature in the presence of buffer, 1 μl MnCl_Twoas well as 1 μl (50 ng) Cleavase^TMThe reaction was started at 55 ° C. by adding the BN enzyme. Les In case 3, MnCl_TwoDenature the DNA in the presence of The reaction was started by the addition of nitrogen. In lane 4, the denaturing mixture was Quality DNA and enzymes, the reaction is performed with buffer and MnCl_TwoAdding To start the reaction. In Lane 5, CFLP^TMBag Fur and MnCl_TwoWas denatured in the presence of, and the enzyme was added at 55 ° C. Leh In step 6, CFLP^TMDenature substrate DNA in the presence of buffer and enzyme, and After MnCl_TwoWas added at 55 ° C. Lane 7 shows the uncleaved control. In lane 8 Is the enzyme and MnCl_TwoDenature the DNA in the presence of Was. In lane 9, the enzyme, MnCl_TwoAnd CFLP^TMSubstrate D in the presence of buffer The NA was denatured and the mixture was incubated at 55 ° C. for 5 minutes. With an "M" The lane containing the biotinylated molecular weight marker produced as described in Example 8. No.   In all cases the reaction was stopped by adding 6 μl of stop buffer . Reaction products (5 μl each) are separated by electrophoresis on a 10% denaturing polyacrylamide gel. The DNA was transferred to a membrane and visualized as described in Example 19. Obtained Oh The radiograph is shown in FIG.   The results shown in FIG. 72 are based on the denaturation initiation protocol used except for the reaction shown in lane 3. This indicates that most of the cols produced the same cleavage pattern. Shown in lane 3 In the reaction, MnCl_TwoIn the presence of CFLP^TMDenatures DNA in the absence of buffer did. Enzyme and MnCl_TwoWas added before the denaturation step (lanes 8, 9), No substance was detected. In these cases, the label is a double-stranded DNA template. The result of nibbling (i.e., terminal nucleotide deletion) of the label from the 5 'end of the plate It was released in the form of short DNA fragments produced as a product.   The results shown in FIG. 72 indicate that the order of addition of the reaction components is 1) MnCl_TwoIn the presence of DNA should not be denatured in the absence of fur solution, and 2) Cleavase^TMBN Enzyme and MnCl_TwoShould not be added simultaneously to the DNA before the denaturation step , Has little effect on the cleavage pattern. These two exceptional articles In some cases, the 5 'label is removed from the 5' end of the substrate by the enzyme, resulting in a loss of signal .                                   Example 29                   Cleavase^TMFragment length polymorphism (CFLP^TM)             Of mutations in the human p53 gene by analysis   The results shown in the previous example show that CFLP^TMReaction of human β-globin and tyrosina Single base changes in fragments of various sizes from the^TM It shows that the reaction can be used to identify different strains of the virus. next , Cleavase detects single base change in human tumor suppressor gene p53^{T M} The ability of the reaction was examined. Mutations in the human p53 gene are the most common tumor-associated residues Gene mutation, with mutations in the p53 gene in about half of all cases of human cancer. Can be seen.   Cleavase^TMThe ability of the BN enzyme to cleave DNA fragments obtained from the human p53 gene, and And the ability to detect single base changes in fragments of the same size. Wild type Or a plasmid containing a cDNA clone containing any of the mutant p53 sequences. Use CFLP^TMA template was prepared for analysis in the reaction. p53 gene Larger, spanning 20,000 base pairs, divided into 11 exons. obtained from cDNA By using a template, it is possible to examine DNA fragments of a given size. The amount of protein coding sequence that can be maximized can be maximized.   SEQ ID NO: 79 shows the nucleotide sequence of the coding region of the wild-type human p53 cDNA gene. Shown in The nucleotide sequence of the coding region of the mutant 143 human p53 cDNA gene This is shown as SEQ ID NO: 80. Mutant 249 (silent) human p53 cDNA gene copy The nucleotide sequence of the nucleotide region is shown as SEQ ID NO: 81. These as follows Generated a 601 nucleotide fragment spanning exons 5-8 from the three p53 cDNAs Was. A) Preparation of substrate DNA   CFLP is used for 6 types of double-stranded substrate DNA^TMPrepared for analysis in the reaction. these The substrate used had a biotin label at the 5 ′ or 3 ′ end. Wild-type substrate A 601 nucleotide fragment spanning exons 5 to 8 of the cDNA sequence of the p53 gene (sequence No. 79) [Baker, S.J. et al., Science (1990) 249: 912]. Two mutations Substrates containing were used. Mutant 143 substrate (SEQ ID NO: 93) was converted from valine (GTG) to From the p53 mutant V143A, which contains a substitution for a single nucleotide (GCG). Leotide changes differ from the wild-type p53 exon 5-8 fragment [Baker, S.J. et al. Science (1990) 249: 912]. Variant 249 (silent) substrate at amino acid 249 Obtained from a p53 mutant containing a single base change from AGG to AGA (SEQ ID NO: 81) . This single base change does not result in a corresponding amino acid change, thus Called silent mutation.   The 601 bp double-stranded PCR fragment was generated as follows. Primer pair 5'-TCTGGGCTTCTTGCATTCTG (SEQ ID NO: 82) and 5'-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO: 8 PCR was started using 3). Synthetic primers are available from Integrated DNA Technologies (C oralville, IA). These primers are available from USB-Amersham (Clevelan d, OH) using the Oligonucleotide Biotinylation Kit purchased from the manufacturer. It was biotinylated at its 5 'end according to the protocol. CFLP sense strand^TMreaction In the case of the analysis in step 5, the 5 ′ end of the primer listed as SEQ ID NO: 82 is Labeled. CFLP^TMWhen analyzing the antisense strand in the reaction, use SEQ ID NO: 83. 5 of the primers listed^'The ends were labeled with biotin.   The tags used in the PCR for the generation of the 601 bp fragment obtained from the wild-type p53 cDNA The target DNA was plasmid CMV-p53-SN3 [Baker, S.J. Et al.], This The plasmid contains the coding region set forth in SEQ ID NO: 79. From mutant 143 The target for the generation of the obtained 601 bp fragment was plasmid CMV-p53-SCX3 [Bake r, S.J. This plasmid contains the coding region set forth in SEQ ID NO: 80, REF. It is a thing. Target for the generation of a 601 bp fragment from mutant 249 (silent) The kit is plasmid LTR 273His [Chen. P.-L. et al., Science (1990) 250: 1576]. This plasmid contains the coding region set forth in SEQ ID NO: 81. DNA is Manufactured from each plasmid carried by the bacteria (plasmid DNA was prepared using standard methods) Isolated). The 601 bp PCR product was produced as follows.   The symmetric PCR reaction was 50 ng of plasmid DNA, 50 pmol of primer 5'-TCTGGGCTTC TTGCATTCTG (SEQ ID NO: 95), 50 pmol of primer 5'-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO: No. 96) and 95 μl of 1 × PCR buffer each containing 50 μM dNTP. reaction Mix 50 μl of ChillOut^TM(MJ Research, Watertown, MA) and tube Was heated to 95 ° C. for 2.5 minutes. Then Taq DNA polymerase (Promega Corp., Mad ison, WI) was added as 1.25 units of enzyme in 5 μl of 1 × PCR buffer. Then heat the tube to 95 ° C for 45 seconds, cool to 55 ° C for 45 seconds, and heat to 72 ° C for 75 seconds 34 cycles and incubate at 72 ° C for 5 minutes after the last cycle did.   The PCR product was gel purified as follows. Final concentration 0.4M NaCl, 20μg glyco The product was precipitated by the addition of a gen carrier and 500 μl of ethanol. Centrifugation Pellet the DNA by separation and equilibrate equal volumes of 95% formamide, 20 mM EDTA and Add solutions containing 0.05% each of lenocyanol and bromophenol blue. The PCR product was resuspended in 25 or 50 μl sterile distilled water. Then heat the tube at 85 ° C Heat for 2 minutes and 6% polyacrylamide with 7M urea in buffer containing 0.5X TBE The reaction products were separated by electrophoresis on a gel (19: 1 cross-link). DNA Visualized by mubromide staining, 0.5M NH_FourSolution containing OAc, 0.1% SDS and 0.1% EDTA Gel-slice the 601 bp fragment by overnight passive diffusion. Cut out from DNA was added to ethanol in the presence of 4 μg of glycogen carrier. By precipitation. Pellet DNA, resuspend in sterile distilled water, Reprecipitation was performed by adding 0.2 M final concentration of NaCl and 80% ethanol. Twice After eye precipitation, pellet the DNA and resuspend in 30 μl sterile distilled water or TE. I let you.   The nucleotide sequences of these 601 bp templates are listed in SEQ ID NOs: 84-89. 6 The sense strand of the 01 nucleotide wild type fragment is listed in SEQ ID NO: 84. 601 nucleotides The antisense strand of the wild-type fragment is set forth in SEQ ID NO: 85. 601 nucleotide variant 143 The sense strand of the fragment is set forth in SEQ ID NO: 86. 601 nucleotide variant 143 fragment anti The sense strand is listed in SEQ ID NO: 87. 601 nucleotide variants of the 249 (silent) fragment The sense strand is listed in SEQ ID NO: 88. 601 nucleotide variants of the 249 (silent) fragment The antisense strand is listed in SEQ ID NO: 89. B) Cleavage reaction conditions   The cleavage reaction is approximately 100 fmole of the resulting duplex in a total volume of 5 μl of sterile distilled water. Substrate DNA (This substrate has a biotin moiety at the 5 'end of the sense or antisense strand. Including). Heat the reaction to 95 ° C for 15 seconds to denature the substrate, then Cooled quickly to 50 ° C (this step reduces the formation of intrachain hydrogen bonds between complementary bases). DNA has a unique secondary structure).   Heated to 95 ° C for 15 seconds, then immediately cooled to 50 ° C Reactions were performed in a mocycler (MJ Research, Watertown, MA).   After cooling the tube to a reaction temperature of 50 ° C, the following components, 0.2 μl of Cleavase^TMBN 5 μl of the diluted enzyme mixture containing [50 g / μl 1X Cleavase^TMBN dilution buffer (0.5% NP 40, 0.5% Tween 20, 20 mM Tris-Cl, pH 8.0, 50 mM KCl, 10 μg / ml BSA]: 1 μl of 1 0X CFLP^TMReaction buffer (100 mM MOPS, pH 7.5, 0.5% NP40, 0.5% Tween20) and 1 μl of 2 mM MnCl_TwoWas added.   A control without enzyme (10 μl) was provided in parallel for each tested PCR fragment. Was. This control is called Cleavase^TMOnly by replacing BN enzyme with sterilized distilled water It was different from the above reaction mixture. After 3 minutes add 8 μl of stop buffer To stop the reaction.   The samples were then heated to 85 ° C for 2 minutes and 4 μl of each reaction mixture was added to 0.5X On a 6% polyacrylamide gel (19: 1 cross-linking) containing 7M urea in a buffer containing TBE Was separated by electrophoresis.   After electrophoresis, the gel plate is separated and the gel remains flat on one plate It was made to become. Positively charged nails with a pore size of 0.2 μm pre-wetted with 0.5X TBE Acrylamide gel with exposed Ron film (Schleicher and Schuell, Keene, NH) Layered. Any bubbles remaining between the gel and the membrane were removed. After that, two 3MM filter papers (Wh atman) on the membrane, replace the other glass plate, and remove the sandwich Clipped with clips. The transfer was overnight. After transfer, carefully remove the membrane from the gel. 1X Sequencase Images Blocking Buffer (United States Biochemical) Washing was performed twice with gentle stirring at 5 minute intervals. Cm of membrane^Two3 / 10ml buffs per Used. Streptavidin-alkaline phosphatase conjugate (SAAP, U nited States Biochemical, Clearland, OH) at 1: 3000 dilution Added directly to the solution and stirred for 15 minutes. Membrane is washed with 1X SAAP buffer containing 0.1% SDS (100 mM Tris-HCl, pH10: 50 mM NaCl)^Two3 times using 0.5ml per 5 minutes (5 minutes / wash It was washed. The membrane was then washed with 1 mM MgCl without SDS._Two1X SAAP buffer containing Was washed twice, drained sufficiently, and placed in a heat-sealable bag. Sterilized pipette Using tip, 0.05ml / cm^TwoCDP-Star^TM(Tropix, Bedford, MA) For 5 minutes. All excess liquid and air bubbles were removed from the bag. Then membrane Exposed to X-ray film (Kodak XRP) for the first 30 minutes. Exposure was performed. Exposure time was adjusted as needed to obtain resolution and lightness. Get The resulting autoradiograph is shown in FIG.   In FIG. 73, the lane labeled M contains a biotinylated molecular weight marker. Ma Maker fragments were purchased from Amersham (Arlington Heights, IL). Lanes 1-4 , Cleavase^TMIncubation of the double-stranded DNA substrate in the absence of the BN enzyme (ie, non- Cleaved control). Lane 1 is the sense of the 601 bp PCR fragment Contains the wild-type fragment labeled on the strand. Lane 2 was labeled with the sense strand of the 601 bp PCR fragment. Mutant 143 fragment. Lane 3 shows wild-type labeled on the antisense strand of the PCR product. Includes type fragments. Lane 4 corresponds to amino acid 249 labeled on the antisense strand of the PCR product. Includes fragments encoding silent mutations in Lanes 5-8 have two 601 bp Clear substrate^TMIncludes reaction products obtained by incubation with BN enzyme. Les Lane 5 contains the product generated using the wild-type fragment labeled on the sense strand, Step 6 contains the product generated using mutant 143 labeled on the sense strand. lane 7 and 8 are wild-type and mutant 249 (silent) substrates labeled on the antisense strand , Respectively.   The results shown in FIG.^TMWild-type and mutant-containing balances with BN enzyme A similar but distinctly different pattern of cleavage products is obtained by It indicates that it was done. By comparing lanes 5 and 6, a range of 100 nucleotides was obtained. The differences in the surrounding band patterns are apparent. Specifically, it exists in the wild type Strong band (around 100 nucleotides) is lost in the V143A mutant, The two bands immediately below the band are prominent in the mutant and clear in the wild type. Absent. In the 200 nucleotide range, the distinctive dub found in wild-type The lett is missing in the mutant and instead is slightly less than the wild-type doublet It contained a strong single band that moved fast. Similarly, comparing lanes 7 and 8 From the antisense strand cleavage of the wild-type and mutant 249 (silent) fragments. The differences between the generated patterns are apparent. In the range of 100 nucleotides Shows that the wild type fragment showed a strong doublet, but the upper band of this doublet It was not found in the mutant 249 (silent) fragment. In addition, 150-180 bp Two dominant bands present in the wild-type pattern in the range are mutant 249 (sa (Irrent) Nothing was found in the cleavage product.   Each mutant fragment analyzed in FIG. 73 contains only one of the 601 nucleotides. Although different from the native form, a unique pattern of cleavage fragments was generated for each. Sa Furthermore, each change in the immediate vicinity of the DNA sequence change (i.e., in the range of 10-20 nucleotides). At least one pattern change has occurred in the variant. In this experiment, CFLP^TMBut p53 remains Can distinguish the presence of a single base change in a PCR fragment containing the exons 5-8 of the gene Which indicates that.                                   Example 30                       In the PCR fragment of the human p53 gene                  Detection of genetically engineered mutations   Genetically engineered into a PCR fragment containing exons 5-8 of the human p53 gene Cleavase that detects single base changes^TMThe ability of BN enzyme was analyzed. Single introduced The base change is 1) a change at amino acid 249 from arginine (AGG) to serine (AGT) ( R249S mutation) and 2) histidine from arginine (CGT) at amino acid 273. Gin (CAT) (referred to as R273H mutation). These mutations They are also found in human tumors and have been identified as mutation hotspots [H ollstein et al., Science 253: 49 (1991)]. The R249S mutation is exposed to aflatoxin B And / or has a strong correlation with hepatitis B virus infection [Ca ron de Fromental and Soussi, Genes, Chromosomes and Cancer (1992) pp. 1-15 ]. The R273H mutation occurs as a result of a change in the CpG dinucleotide. So Such changes amount to about one-third of known p53 mutations and are characteristic of various tumor types. [Caron de Fromental and Soussi, supra: Hollstein et al., Supra].   Plasmids containing the R249S and R273H mutations were Higuchi [PCR Technology: Pri nciple and Applications for DNA Amplificarion, H.A. Ehrlich, Ed. (1989) St ockton Press. NY. pp. 61-70] Manufactured. By this method, the DNA sequence corresponding to the known p53 mutation is included A collection of plasmids can be produced. This collection is available By that, Cleavase^TMCFLP generated by cleavage of p53 mutants using enzymes^T A p53 "barcode" library containing the M pattern is generated. A) Construction of a 601 bp PCR fragment containing the R249S mutation   A two-stage recombinant PCR was performed to generate a 601 bp fragment containing the R249S mutation. (See FIG. 6 for an overview of this two-step recombinant PCR.) First, or "on In the `` flow '' PCR, the oligonucleotide 5'-TCTGGGCTTCTTGCATTCTG (SEQ ID NO: 82) And use the 5'-GAGGATGGGACTCCGGTTCATG (SEQ ID NO: 90) to generate the R249S mutation A 427 bp fragment containing a base change from G to T was amplified. The sequence of this 427 bp fragment is No. 98. The second, or "downstream" PCR is oligonucleotide 5'-CATGAACC Using GGAGTCCCATCCTCAC (SEQ ID NO: 91) and 5'-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO: 83) A 196 bp fragment containing the same base change in the complementary strand was used for amplification. Sequence of this 196 bp fragment Is listed in SEQ ID NO: 99.   For each PCR, a cDNA clone encoding the wild-type p53 gene (SEQ ID NO: 79) 10 ng of the coding region (see above) as a template in a 50 μl PCR reaction. Was. For upstream fragments, 5 pmole of oligonucleotide in 45 μl 1X PCR buffer Tide 5'-TCTGGGCTTCTTGCATT CTG (SEQ ID NO: 82), 5pmole of oligonucleotide 5 ' -10 g in a tube containing GAGGATGGGACTCC GGTTCATG (SEQ ID NO: 90) and 50 μM of each dNTP g template was added. For downstream fragments, 5 pmole in 1X PCR buffer Oligonucleotides 5'-CATGAACCGGAGTCCCATCCTCAC (SEQ ID NO: 91) and 5pmole of Oligonucleotide 5'-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO: 83) and 50 μM of each dNTP , 10 ng of wild-type template, plasmid CMV-p53-SN3 (Example 29).   50 μl in a tube containing 45 μl of the above mixture for each template to be amplified ChillOut^TM(MJ Research, Watertown, MA) and place the tube at 95 ° C for 2.5 minutes. And then cooled to 70 ° C. 1X PCR with Taq DNA polymerase (Promega) Added as 1.25 units of enzyme in 5 μl of buffer. Then for 24 cycles Heat the tube to 95 ° C for 45 seconds, cool to 55 ° C for 45 seconds, and heat to 72 ° C for 75 seconds And incubated at 72 ° C. for 5 minutes after the last cycle.   The PCR product was gel purified as follows. Add 10 μl of each PCR product to 10 μl Mix with stop buffer. Then heat the tube at 85 ° C for 2 minutes, containing 0.5X TBE Electrophoresis on 6% polyacrylamide gel (7: 1 cross-link) containing 7M urea in buffer (The used polyacrylamide gel solution was newly prepared.) Made). DNA was visualized by ethidium bromide staining and 0.5M NH_FourOAc, 0.1 Passive diffusion at 37 ° C into a solution containing% SDS and 0.1% EDTA The fragments were cut out of the gel slice by running overnight.   A second round of 10 μl of each eluted PCR product was used as recombinant template and used as a recombinant template. PCR was started. This template was added to 10 pmole of 5'-vial in 1X PCR buffer. Otin exon 8 primer (SEQ ID NO: 83), 10 pmole of 5'-exon 5 primer -(SEQ ID NO: 82) and 50 μM of each dNTP were added. For each template to be amplified 50 μl ChillOut into a tube containing 90 μl of the above mixture^TM(MJ Research, Wate rtown, MA) and heated the tube to 95 ° C for 2.5 minutes, then cooled to 70 ° C . 2.5 units of Taq DNA polymerase (Promega) in 5 μl of 1X PCR buffer Was added as an enzyme. Then heat the tube to 95 ° C for 45 seconds, cool to 47 ° C Anneal the two template molecules, then heat to 72 ° C to The primer was extended by merase. The beginning of this denaturation, annealing and extension After one cycle, the reaction was heated to 95 ° C. for 45 seconds, cooled to 55 ° C. for 45 seconds, and cooled to 72 ° C. for 1 second. The heating cycle for 25 minutes was performed 25 times, and then extended at 72 ° C. for 5 minutes. afterwards Fragments were ethanol precipitated and gel purified as described in Example 29. B) Construction of a 601 bp PCR fragment containing the R273H mutation   Use the procedure described in a) to generate a 601 bp fragment containing the R273H mutation. A two-stage recombinant PCR was performed using amino acid 273 to convert arginine (CGT) to amino acid 273. A PCR fragment encoding a single base change to stidine (CAT) was co-amplified. the first Alternatively, in "upstream" PCR, the oligonucleotide 5'-TCTGGGCTTCTTGCATTC Using TG-3 ′ (SEQ ID NO: 82) and 5′-GCACAAACATGCACCTCAAAGCT-3 ′ (SEQ ID NO: 92) The 498 bp fragment listed in SEQ ID NO: 100 was generated. In a second or “downstream” PCR And the oligonucleotide 5'-CAGCTTTGAGGTGCATGTTTGT-3 '(SEQ ID NO: 93) Paired with nucleotide 5'-GTTGGGCAGTGCTCGCTTAG-3 '(SEQ ID NO: 83) To generate the 127 nucleotide fragment listed in SEQ ID NO: 101. These DNA fragments Electrophorese, elute and combine for a second PCR as described in a). Starting off, a 601 bp PCR product containing the R273H mutation was generated. C) Sequence analysis of 601 nucleotide PCR fragment   The recombinant 601 bp PCR product generated from this two-step PCR procedure is described in Example 29. Used with the oligonucleotide 5'-biotin-GTTGGGCAGTGCTCGCTTAG (SEQ ID NO: 83). PCR products were sequenced and formed according to standard protocols by the manufacturer. The presence of the mutation was confirmed.   The nucleotide sequence corresponding to the sense strand of the 601 nucleotide R249S mutant fragment is Column number 94 lists. The antisense strand of the 601 nucleotide R249S mutant fragment is SEQ ID NO: No. 95. The sense strand of the 601 nucleotide R273H variant fragment is set forth in SEQ ID NO: 96. You. The antisense strand of the 601 nucleotide R273H variant fragment is set forth in SEQ ID NO: 97. D) Cleavage reaction   Subsequent CFLP^TMAdditional PCR rounds to generate large amounts of DNA for analysis Return the 601 bp fragment containing either the R249S or R273H mutation as the template. Used. Approximately 2 fmole of each 601 bp fragment was replaced with SEQ ID NO: 82 and 83 (SEQ ID NO: No. 83 contains biotin at the 5 'end) and 20 pmole of the primer corresponding to dNTP. 50 μM, 20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl, respectively_Two, 50 mM KCl, 0.05% Tween20 and And 0.05% NP40. Tubes containing 90 μl of the above mixture were added to each amplification. Prepare the template to be prepared and add 50 μl of ChillOut to each tube.^TM(MJ Research , Watertown, MA), and heat the tube to 95 ° C for 2.5 minutes, then cool to 70 ° C. Rejected. Then add Taq DNA polymerase (Promega) in 5 μl of 1X PCR buffer. 2.5 units of enzyme. Then heat the tube to 95 ° C for 45 seconds, Cool to 2 ° C. to anneal the two template molecules, then heat to 72 ° C. The primer was extended by aq DNA polymerase. This denaturation, annealing And after the first cycle of extension, heat the reaction to 95 ° C for 45 seconds and cool to 55 ° C for 45 seconds. 25 cycles of heating to 72 ° C for 1 minute, followed by 72 Extended for 5 minutes at ° C. The fragments were then ethanol precipitated as described in Example 29. And gel purified. Thereafter, the gel-purified fragment was subjected to CFLP as follows.^TMUsed for reaction Used.   Cleavage reaction was a total volume of 5 μl (sterilized distilled water was used to bring the volume to 5 μl) About 100 fmoles of the resulting double-stranded substrate DNA (this substrate is (Including a biotin moiety at the 5 'end of the sense strand). Bring reaction to 95 ° C for 15 seconds Heat to denature the substrate and then quickly cool to 50 ° C (this step allows complementary The formation of intrachain hydrogen bonds between the target bases allows the DNA to adopt a unique secondary structure). The reaction is programmed to heat to 95 ° C for 15 seconds and then immediately cool to 50 ° C. In a thermocycler (MJ Research, Watertown, MA) or make a tube manually On a heat block set at 95 ° C, and then heat block set at 50 ° C. Moved on the lock and went.   After cooling the tube to a reaction temperature of 50 ° C, 0.2 μl of Cleavase^TM5 including BN enzyme μl of diluted enzyme mixture [50 ng / μl 1X Cleavase^TMDilution buffer (0.5% NP40, 0.5%  Tween 20, 20 mM Tris-Cl, pH 8.0, 50 mM KCl, 10 μg / ml BSA)], 1 μl of 10X CFLP^TM Reaction buffer (100 mM MOPS, pH 7.5, 0.5% NP40, 0.5% Tween20) and 1 μl of 2m M MnCl_TwoWas added. 10 enzyme-free in parallel for each tested PCR fragment μl of control was provided. This control is called Cleavase^TMBN enzyme replaced with sterile distilled water Was something. After 2 minutes at 50 ° C, add 8 μl of stop buffer to Stopped responding.   Heat the sample to 85 ° C for 2 minutes and dispense 7 μl of each reaction into a bag containing 0.5X TBE. Electrophoresis on 10% polyacrylamide gel (7: 1 cross-link) containing 7M urea in buffer Separated.   After electrophoresis, the gel plate is separated and the gel remains flat on one plate It was made to become. Positively charged nails with a pore size of 0.2 μm pre-wetted with 0.5X TBE Acrylamide gel with exposed Ron film (Schleicher and Schuell, Keene, NH) Layered. Any bubbles remaining between the gel and the membrane were removed. After that, two 3MM filter papers (Wh atman) on the membrane, replace the other glass plate, and remove the sandwich Clipped with clips. The transfer was overnight. After transfer, carefully remove the membrane from the gel. To 1X Sequencase Images Blocking Buffer (United States Biochemical) Washing was performed twice with gentle stirring at intervals of 15 minutes. Cm of membrane^Two3 / 10ml per Buffer was used. Streptavidin-alkaline phosphatase conjugate (S AAP, United States Biochemical, Cleaveland, OH) at 1: 3000 dilution. Added directly to the King solution and stirred for 15 minutes. 1X SAAP buffer with 0.1% SDS for membrane (100 mM Tris-HCl, pH 10: 50 mM NaCl)^Two3 times using 0.5ml per 5 times (5 Min / wash). The membrane was then washed without SDS but with 1 mM MgCl_Two1X SAAP buffer including Washed twice with water, dried sufficiently, and placed in a heat sealable bag. Sterilization Using a pet tip, 0.05ml / cm^TwoCDP-Star^TM(Tropix, Bedford, MA) And allowed to spread over the membrane for 5 minutes. All excess liquid and air bubbles were removed from the bag. That The posterior membrane was exposed to X-ray film (Kodak XRP) for the first 30 minutes of exposure. Exposure Time was adjusted as needed to obtain resolution and lightness. The results are shown in FIG.   In FIG. 74, the lane labeled M contains a biotinylated molecular weight marker. Ma Marker fragments were purchased from Amersham (Arlington Heights, IL), Contains bands corresponding to 00, 200, 300, 400, 500, 700 and 1000 nucleotides in length Things. Lanes 1-4 are Cleavase^TMAntisense strand in the absence of BN enzyme Includes reaction products obtained from incubation of double-stranded DNA substrate labeled . Lane 1 contains the reaction product from the wild-type fragment (SEQ ID NO: 85). Lane 2 is shaped Includes the reaction product from the resulting R249S mutation (SEQ ID NO: 95). Lane 3 is 249 ( (Silent) mutation (SEQ ID NO: 89). Lane 4 is formed Reaction product from the modified R273H mutation (SEQ ID NO: 97).   Lanes 5 to 8 are Cleavase^TMThis when incubated in the presence of the BN enzyme And the cleavage products generated from the respective sense strands of these templates. Leh Step 5 contains the cleavage product from the wild-type fragment (SEQ ID NO: 84). Lane 6 is R249S cut Fragment (SEQ ID NO: 94). Lane 7 is 249 (silent) mutant Includes the cleavage product from the fragment (SEQ ID NO: 88). Lane 8 shows the R273S fragment (SEQ ID NO: 9). 6).   The results shown in Figure 74 show that each of these templates containing a single base change Indicates that a similar but distinctly different pattern of cleavage was generated. ing. Lane 6 shows 15% when compared to the wild type pattern shown in Lane 5. Bands in the range 0-180 nucleotides are weakened and bands in the 100 nucleotide range It shows that it disappears. In addition, lane 6 contains a new bus in the 140 nucleotide range. And increased intensity of the upper band of the doublet, about 120 nucleotides. doing. A different nucleotide from the wild type at the same nucleotide position as R249S (lane 6). Examination of the Irent 249 mutant (lane 7) revealed that the wild type (lane 5) as well as R249S (lane 6). ) Pattern differences for both mutations were revealed. Specifically, When compared to Lane 5, as seen in Lane 6, the range of 150-180 nucleotides Band is weakened, indicating that bands in the 100 nucleotide range disappear. . However, the sample in lane 7 did not show another band in the 140 nucleotide range, Of the upper band of the doublet in the range of 120 nucleotides seen in region 6 Also not shown. This result is^TMIf different salts at the same nucleotide position It indicates that the group changes can be distinguished.   When the reaction product in lane 8 was examined, it was compared with the wild type pattern shown in lane 5. As a result, it was determined that the band in the 100 nucleotide range in the R273S fragment had disappeared. You. But this CFLP^TMPattern weakened band in the range of 150-180 nucleotides Is different from that of lanes 6 and 7 in that no In the region, this pattern is virtually indistinguishable from that produced from wild-type fragments. Absent.   The above results indicate that CFL was used to detect clinically significant mutations in human p53. P^TMIndicates that can be used. Furthermore, these results indicate that CFLP^TMLaw is the same Sufficient to distinguish different base changes in the position from each other and from the wild type. It shows that it has sensitivity. These results also indicate that the known p53 mutation Shows that the 2-PCR method can be used to produce a collection of PCR fragments containing I have. With such a collection, CFLs generated by different p53 mutations P^TMA p53 barcode library containing patterns can be generated.                                   Example 31              Detection of the presence of wild-type and mutant sequences in mixed samples   Different alleles of the same size PCR fragment in a mixed sample, e.g., heterologous Detect presence of conjugates or heterogeneous tissue, such as can be found in samples CFLP to do^TMThe ability of the reaction was examined.   For wild-type p53 (SEQ ID NO: 84) and mutant 143 (SEQ ID NO: 86) 601-bp fragments, A PCR product containing a biotin label on the sense strand was prepared as described in Example 29. And purified. Aliquots of these samples were made to a final concentration of about 12.5 fmole / μl And mix at different ratios to give a ratio of wild-type to mutant DNA. Got a toll. A total of about 50 fmole of D in each sample mixed in various combinations For NA, place 4 μl of diluted DNA sample into microfuge tube , Heated at 95 ° C. for 15 seconds. The tube was rapidly cooled to 50 ° C and 0.2 μl of Cleavase^TMBN Dilute enzyme mixture containing enzyme [50 ng / μl 1X Cleavase^TMDilution buffer (0.5% NP40 , 0.5% Tween 20, 20 mM Tris-Cl, pH 8.0, 50 mM KCl, 10 μg / ml BSA)] μl 10X CFLP^TMReaction buffer (100 mM MOPS, pH 7.5, 0.5% NP40, 0.5% Tween20) And 1 μl of 2 mM MnCl_TwoWas added. Cleavase^TMUse sterile distilled water instead of BN enzyme Were used in parallel for each PCR fragment tested, with 10 μl of enzyme-free control I thought. After 1.5 minutes at 50 ° C., the reaction was stopped by adding 8 μl of stop buffer. Sa Furthermore, for comparison with the mixed sample, only 4 μl of wild type and only 4 μl of V143A Was analyzed in a similar manner.   The sample was heated at 85 ° C for 2 minutes, containing 7M urea in buffer containing 0.5X TBE. 7 μl of the reaction was separated by electrophoresis on a 10% polyacrylimide gel (19: 1 cross-linking). Released.   After electrophoresis, release the gel plate and leave the gel flat on one plate. It was to so. A positively charged nylon membrane with a pore diameter of 0.2 μm (S chleicher and Schuell, Keene NH) were overlaid on the exposed acrylamide gel. All air bubbles between the gel and the membrane were removed. Place two 3MM filter papers (Whatman) on the membrane Then, the other glass plate was placed again, and the sandwich was clipped. Transfer Movement was performed overnight. After transfer, carefully remove the membrane from the gel and gently agitate. 15 minutes in 1X Sequencase Inages Blocking Buffer (United States Biochmical) Washed twice at intervals. 1cm membrane^TwoUsed a 3/10 ml buffer. Strike Leptoavidin-alkaline phosphatase conjugate (SAAP, United States Bioch) emical, Cleaveland, OH) was added directly to the blocking solution to a dilution of 1: 3000 and 15 Stirred for minutes. The membrane was washed with 1X SAAP buffer (100 mM Tris-HCl, pH 1cm membrane in 10,50mM NaCl)^TwoWashes three times (5 minutes / wash) using 0.5 ml per wash. Next The membrane does not contain SDS but 1mM MgCl_TwoWash twice with 1X SAAP buffer containing Drained in minutes and placed in heat sealable bags. Use a sterile pipette tip And 0.05ml / cm^TwoCDP-Star^TM(Tropix, Bedford, MA) and add to bag for 5 minutes Let it go. All excess liquid and air bubbles were removed from the bag. After that, the film was converted to X-ray film (K (odak XRP) for the first 30 minutes. Exposure time depends on resolution and brightness Adjusted as needed to get. The obtained autoradiogram is shown in FIG.   In FIG. 75, the lane labeled M contains a biotinylated molecular weight marker. Ma Makers were obtained from Amersham (Arlington Heights, IL), 50, 100, Containing bands corresponding to 200, 300, 400, 500, 700 and 1000 nucleotides in length It is. Lanes 1 and 2 show the enzymes for wild-type and V143A mutant fragments, respectively. It contains the reaction products from the control without the element. Lane 3 is wild type fragment only Contains cleavage products from samples containing Lane 4 is mixed at a 1: 1 ratio It contains cleavage products from isolated wild-type and mutant fragments. Lane 5 is wild type And cleavage products from reactions containing the mutant fragments in a ratio of 1: 2. lane 6 contains the reaction product in which the wild type and mutant fragments are present in a ratio of 1: 9. Les Region 7 contains cleavage products from a sample containing only V143A mutant DNA . Lane 8 contains cleavage products of wild type and mutant mixed at a ratio of 2: 1. I have. Lane 9 contains cleavage products of wild type and mutant mixed at a ratio of 4: 1. In. Lane 10 contains the cleavage product of the wild type and the mutant mixed at a ratio of 9: 1. It is.   The results shown in Figure 75 demonstrate that the presence of different alleles can be detected in mixed samples. Are shown. Compare lanes 4-6 and lanes 8-10 with lane 3 or lane 7. By comparison, the lane containing the mixed reactants has a discernible difference from either sample alone. It turns out that it shows a difference. Especially in the 100 nucleotide region, the mutant shifts The doublet is in the wild type sample (see discussion of FIG. 74 in Example 29). Lane 3 and And 7 could detect only one or the other pair, whereas the mixed sample All three of these bands are present in lanes (lanes 4-6 and lanes 8-10).                                   Example 32                     Cleavase^TMBy fragment length polymorphism analysis                   Detection and identification of hepatitis C virus genotype   Hepatitis C virus (HCV) infection is the leading type of post-transfusion non-A non-B (NANB) hepatitis worldwide. Cause. In addition, HCV is a major global source of hepatocellular carcinoma (HCC) and chronic liver disease. It is an important pathogen. For molecular biological analysis of small (9.4kb) RNA genome Some regions of the genome are more likely to mutate more rapidly than others, It has been shown to be very highly conserved between isolates. In this analysis, Viruses were divided into six basic genotype groups and further subdivided into several subtypes. [Altamirano et al. Infect. Dis. 171: 1034 (1995)]. These viruses Groups are associated with different geographical areas, and in disease surveillance, It is important to accurately identify the substance. In the United States, genotype 1 HCV , But multiple HCV genotypes have been found in both Europe and Japan. I have. HCV genotype is also associated with differences in the efficacy of treatment with interferon. Individuals infected with group 1 show little response. To infected individuals If an existing HCV genotype can be identified, infection from different types of HCV Clinical outcomes from infection with multiple types of HCV in a single individual Comparison becomes possible. By pre-screening infected individuals for virus types, Clinicians can make more accurate diagnoses, and are more costly and ineffective Treatment can be avoided.   To develop a quick and accurate method for typing HCV present in infected individuals To, Cleavase^TMThe reaction identifies and detects major HCV genotypes and subtypes. I checked whether it could be done. Conserved 5 'of six different HCV RNA isolates Using a plasmid containing DNA obtained from the untranslated region, CFLP^TMFor analysis in reactions Generated template. The HCV sequences contained in these six plasmids are: Genotype 1 (4 subtypes represented by 1a, 1b, 1c and Δ1), 2 and 3 It is something to represent. The HCV genotype nomenclature used was that of Simmonds et al. (Described in Altamirano et al., Supra). A) Generation of plasmid containing HCV sequence   Using RNA extracted from blood donor serum samples, RT-PCR Six types of DNA fragments obtained from HCV were generated. These PCR fragments were Dr. Altamirano (University of British Columbia, Vancouver). This These PCR fragments contained HCV sequences obtained from HCV genotypes 1a, 1b, 1c, Δ1c, 2c, and 3a. To represent.   RNA extraction, reverse transcription and PCR are performed using standard techniques [Altamirano et al. Infect. Dis . 171: 1034 (1995)]. Briefly, guanidine isothio Using cyanate, sodium lauryl sarcosinate and phenol chloroform RNA was extracted from 100 μl of serum [Inchauspe et al., Hepatology 14: 595 (1991). )]. Reverse transcription was performed using GeneAmprTh reverse in the presence of the external antisense primer HCV342. Performed using transcriptase RNA PCR kit (Perkin-Elmer) according to manufacturer's instructions . The sequence of the HCV342 primer is 5'-GGTTTTTCTTTGAGGTTTAG-3 '(SEQ ID NO: 102). You. After completion of the RT reaction, the sense primer HCV7 [5'-GCGACACTCCACCATAGAT-3 '(sequence No. 103)] and magnesium, and the first PCR was performed. First PCR product Recoat the primers HCV46 [5'-CTGTCTTCACGCAGAAAGC-3 ') (SEQ ID NO: 104) and In the presence of HCV308 [5'-GCACGGTCTACGAGACCTC-3 ') (SEQ ID NO: 105), a second ( ) Was used for PCR. These PCRs allow the region of HCV to be located between positions 284 and 4 of the HCV genome. A 281 bp product corresponding to the conserved 5 'non-coding region (NCR) was generated [Altam irano et al. Infect. Dis. 171: 1034 (1995)].   Six 281 bp PCR fragments can be used directly for cloning or used for additional amplification steps I took it. At this stage, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl_TwoAnd 0. In a buffer containing 1% Tween20, about 10 fmoles of DNA, each 0.1 μM HCV46 And HCV308 primers, 100 μM all four dNTPs and 2.5 units of Taq polymer 50 μl of PCR containing the enzyme was used. PCR was performed at 96 ° C for 45 seconds, 55 The cycle was 45 times at 45 ° C. and 1 minute at 72 ° C. 25 times. Original DNA sample or Was performed using 2 μl of the reamplified PCR product according to the manufacturer's protocol according to the manufacturer's protocol. Cloned into lue T-vector (Novagen, Madison, WI). PCR products to pT7 After ligation to the Blue T-vector, the ligation mixture is used to competent JM109 cells. Vesicles (Promega) were transformed. Clos containing pT7Blue T-vector with insert LB containing 40 μg / ml X-Gal, 40 μg / ml IPTG and 50 μg / ml ampicillin Selection was made on the plate by the presence of white colonies. About each PCR sample Four colonies were picked and overnight in 2 ml of LB medium containing 50 μg / ml carbenicillin Propagated. Plasmid DNA using the alkaline miniprep protocol described below Was isolated. Cells from 1.5 ml of overnight culture were removed in a microcentrifuge (14K rpm) for 2 minutes The supernatant was discarded and the cell pellet was washed with 10 μg / ml RNase A (Phar macia) in 50 μl of TE buffer. Solution containing 0.2N NaOH, 1% SDS 100 μl of the solution was added and the cells were lysed for 2 minutes. Lysate 100 μl of 1.32 M potassium acetate Gently mix with pH 4.8 and centrifuge the mixture in a microcentrifuge (14K rpm) for 4 minutes Separated and discarded pellet containing cell debris. 200 μl of plasmid DNA Precipitate the supernatant with ethanol and pellet by microcentrifugation (14K rpm) did. The DNA pellet was air dried for 15 minutes and then redissolved in 50 μl of TE.   30 cycles of amplification were used to analyze the cloned HCV insert Except for the above, using the primers HVCV46 and HCV308, Mid DNA (about 10 to 100 ng) was reamplified in 50 μl of PCR. 0.5X TBE buffer PCR products by electrophoresis on a 6% non-denaturing acrylamide gel (29: 1 cross-linking) Clones that were separated and yielded a 281 bp PCR product were selected for further analysis.   For sequencing, plasmid DNA from selected clones was Was purified by PEG. 50 μl of plasmid DNA (about 10-100 ng / μl) in TE buffer , 25 μl 5 M NaCl and 10 μl 20% PEG (M.W. 8,000: Fisher) and mix well The mixture was incubated on ice for 1 hour. The mixture is then placed on a tabletop microcentrifuge. Centrifuge by centrifugation (14K rpm) for 5 minutes, remove the pellet, and add another 20% 15 μl of PEG was added. After incubating on ice for 1 hour, centrifuge the second pellet Collect by separation, discard supernatant and pellet 20 μl H_TwoRedissolved in O. PEG purification 2 μl of plasmid DNA (about 100 ng) was used for HCV46 or HCV308 primer. Used for cycle sequencing reactions according to the manufacturer's protocol. HCV46 or HCV30 8 Primers should be used before Oligonucleotide Biotin Labeling The 5 'end was biotinylated using a kit (Amersham, Arlington Heights, IL) . Sequencing reactions were separated on a 10% denaturing acrylamide gel and transferred to a nylon membrane. And visualized as described in Example 19.   Alternatively, the 5 'end is tetrachlorofluorescein (TET) dye (Integrated  DNA Technologies) -labeled Blue-T1 [5'-GATCTACTAGTCATATGGAT-3 ') (SEQ ID NO: 106)] and Blue-T2 [5'-TCGGTACCCGGGGATCCGAT-3 ') (SEQ ID NO: 107)] And DNA sequencing was performed. In this case, the sequencing reaction was 10% denatured acrylic Separated on an amide gel and the product was analyzed using an FMBIO-100 Image Analyzer (Hitachi). Visualized. The six HCV clones are HCV1.1, HCH2.1, HCV3.1, HCV4.2, HCV6.1 And HCV 7.1, and the double-stranded DNA sequences of these clones are represented by SEQ ID NO: 108-113. The sequence of the sense strand of each of the six HCV clones is SEQ ID NO: 1. Shown in the top row of 08-113. HCV clone, HCV1.1, HCH2.1, HCV3.1, HCV4.2, HCV6 The sequences of the antisense strand of .1 and HCV7.1 are listed in SEQ ID NOS: 114-118, respectively.   The DNA sequences of each of the six HCV clones are listed in FIG. In FIG. 76, Nucleotides representing changes between the six HCV clones are in bold and underlined. Show. Dash to indicate gaps introduced to maximize alignment between sequences (Required for insertion found in clone HCV4.2). This alignment is Show that these six HCV clones represent six different genotypes. H CV1.1 represents genotype 1c HCV, HCV2.1 represents genotype 1a HCV, HCV3.1 represents genetic HCV4.2 represents genotype 1c HCV, HCV6.1 represents genotype 2c HCV, HCV 7.1 represents genotype 3a HCV. One sample, HCV4.2 , An insertion of the “G” nucleotide is found at position 146 (for proprietary HCV T: Altamirano et al., Supra), that insertions and deletions have been reported in the HCV NCR. No second independent clone obtained from the PCR product corresponding to HCV4 was sequenced. Was decided. This second HCV4 clone has the same arrangement as shown in FIG. 76 for HCV4.2. Was found to have columns. B) Preparation of HCV substrate   CFLP^TMSix double-stranded substrate DNAs were prepared for analysis in the reaction. This substrate The use of labeled primers during PCR allows sense and antisense The 5 'end of either strand is labeled, and each strand of the HCV DNA substrate is^TMAnalyze Can be otide Phosphate Labeling Kit (Molecular Probes. Inc., Eugene. OR) Follow the manufacturer's protocol with the TMR dye at the 5 'end of the HCV46 and HCV308 primers. Labeled. All six HCV 281 bp NCR sequences were a) using 10 ng of template PCR amplification was performed with 30 cycles of amplification, as described in.   For sense strand analysis, TMR labeled HCV46 primer (SEQ ID NO: 104) PCR was performed using the unlabeled HCV308 primer (SEQ ID NO: 105). A For analysis of the antisense strand, the unlabeled HCV46 primer (SEQ ID NO: 10 PCR was performed using 4) and the HCV308 primer (SEQ ID NO: 105) labeled with TMR. PCR The product was electrophoresed on a 6% denaturing acrylamide gel as described in Example 19. Purified by kinetics and eluted overnight. Gel purified DNA substrate was mixed with 20 μl of H_TwoConcentration about 10 in O Redissolved in 0 fmole / μl. C) Cleavage reaction conditions   Cleavage reactions were 0.5% Tween 20 and NP-40TMR and 10 ng Cleavase, respectively.^TMBN yeast 1 μl of TMR-labeled PCR product (about 100 fm) in a total of 10 μl of 10 nM MOPS, pH 7.5, ole double-stranded substrate). MnCl_TwoAll components except for 8 μl volume I combined. Heat the reaction to 95 ° C for 15 seconds to denature the substrate, then quickly cool to 55 ° C Rejected. The reaction was heated to 95 ° C for 15 seconds and then immediately cooled to 55 ° C. In a simulated thermocycler (MJ Research, Watertown, MA) or The heat block manually set on a heat block set to 95 ° C and then to 55 ° C. And moved on the second heat block set in the above.   After cooling the tube to the reaction temperature of 55 ° C, 2 μl of 1 mM MnCl_TwoAdding Initiates the cleavage reaction. After 2 minutes at 55 ° C, 95% formamide, 10 mM EDTA and Stop the reaction by adding 5 μl of a solution containing 0.02% methyl violet did.   Heat 5 μl of each reaction mixture at 85 ° C. for 2 minutes, and add 7M urine in 0.5 × TBE buffer. Electrophoresis on a 12% denaturing polyacrylamide gel containing separated. The gel was run at 33 watts for 1.5 hours. FMBIO labeled reaction products Visualization was performed using a -100 Image Analyzer (Hitachi). Obtained fluoro image The flash scan is shown in FIG.   In FIG. 77, production by cleavage of six HCV samples labeled on the sense strand CFLP generated^TMThe patterns are shown in lanes 1-6. Labeled on antisense strand CFLP generated by cleavage of six HCV samples^TMThe pattern is from lanes 7 to 12 Shown in The position of the molecular weight marker is large on the left side of the fluoroimager scan. Indicated by a simple arrow. Marker sizes are indicated in nucleotides.   The experiment shown in Figure 77 shows that CFLP distinguishes six distinct hepatitis C viruses.^TM The ability is shown. The six samples on the left side of the panel (lanes 1-6) are The 5 ′ end of the antisense strand is labeled, and the right six (lanes 7-12) are labeled The ends were labeled. Are the first four samples in each set all HCV type 1? Including amplified samples. Subtypes a, b and c are of type 1c (i.e., Δ1c) As a single base deletion. From the analysis of each chain, many classes between subtypes Similarities and some distinct differences are noted. Most similarity in the sense strand Also prominent are the dominant bands shown as A, B and C. In particular, Bands B and C are evident in patterns generated from both subtypes 1a and 1b. While (and in fact are more dominant in subtype 1a than subtype 1a) ), It can hardly be seen in subtype 1c. Band A uses these samples Are present in all four, but in patterns generated from subtypes 1c and 1a More dominant. Difference between subtypes 2c and 3a and all of the subtype 1 samples The difference is in the region between 50 nt and 100 nt of the sense strand (compare bands D and E). And the region between 80 nt and 150 nt of the antisense strand (compare bands FJ) Is clear. Virus type 3 looks similar to type 1, but type 2 is the most Significantly changed CFLP^TMIndicates a pattern. This relationship is a consequence of sequence differences between different isolates. Seems to be consistent with the relative number of differences.   From the results shown in FIG. 77, CFLP^TMSimpler and faster method to determine genotype of HCV strain It has been shown to provide a law. This method facilitates the diagnosis of HCV infection and And help monitor the development of HCV.                                   Example 33                   Mycobacterium tuberculosis               Of mutations associated with antibiotic resistance in rice   The last decade has seen a tremendous resurgence of tuberculosis in its home country and around the world. Global scale Thus, the number of new cases reported per year increased from 7.5 million in 1990 to 102 in 2000. It is expected to increase to 100,000. One feature that warns of the resurgence of TB is one or more. M. tuberculosis strains resistant to the above anti-tuberculosis drugs [ie, multiple drug resistant tuberculosis (MDR-TB)] It is an increase in the number of patients.   Antibiotic rifampin (rif) and / or isoniazid (inh) Resistance to is a standard to determine whether a Mycobacterium tuberculosis strain is multi-drug resistant. It is rare to obtain their strong bactericidal action and primary resistance to these drugs. (The spontaneous mutation rate of rifampin resistance is about 10^-8, Isoniazid resistant Then 10^-8From 10^-9), Until recently, these two antibiotics have developed and spread It was the most powerful frontline drug used to eradicate the nucleus. But tuberculosis in the United States In a patient survey, as many as one-third were infected with one or more anti-TB drugs More than 25% of M. tuberculosis cultures isolated are isoniazid resistant and 9% were found to be resistant to both isoniazid and rifampin [Frieden et al. , New Eng. J. Med., 328: 521 (1993)].   As explained above (Description of the invention), rifampin resistance is the rpoB gene of M. tuberculosis. Is associated with a mutation. The exact mechanism of isoniazid resistance is not clear, inh^rMost mutations (up to 80%) occur in the katG and inhA genes of M. tuberculosis. CFLP^TMCan be used to detect mutations in genes involved in MDR-TB? DNA fragment was amplified from the rpoB and katG genes of M. tuberculosis . Wild-type (i.e., antibiotic-sensitive) or mutant (i.e., antibiotic-resistant) strain of M. tuberculosis DNA fragment obtained from^TManalyzed. A) CFLP of a mutation in the rpoB gene of Mycobacterium tuberculosis^TManalysis i) Generation of plasmid containing rpoB gene sequence   Contains mutations in the rpoB gene associated with wild-type M. tuberculosis or rifampin resistance Genomic DNA isolated from M. tuberculosis strains is Dr. Schinnick (Centers for Dis ease Control and Prevention, Atlanta, GA). Rifampin resistant Strain # 13 (91-3083) contains the histidine found in the wild-type strain at codon 451 of the rpoB gene. It contains a tyrosine residue instead of a residue (ie H451Y). This mutation Present in 28% of ampin-resistant TB isolates. The H451Y mutation is referred to below as Mutant 1. You. Rifampin-resistant strain # 56 (91-2763) has a wild-type strain at codon 456 of the rpoB gene. Contains a leucine residue in place of the serine residue found in (S456L). this The mutation is present in 52% of rifampin-resistant TB isolates. The S456L mutation is It is referred to as mutant 2.   From DNA obtained from wild-type, mutant 1 and mutant 2 strains, TB rpoB The 620 bp region of the gene was amplified. The ply used to amplify the rpoB gene sequence The mers are PolB-5A [5'-ATCAACATCCGGCCGGTGGT-3 '(SEQ ID NO: 120)] and PolB-5B [5'-G GGGCCTCGCTACGGACCAG-3 ′ (SEQ ID NO: 121)], and these PCR primers are H451Y Amplify a 620 bp region of the rpoB gene spanning both the S456L and S456L mutations [Miller et al., A ntimicrob, Agents Chemother., 38: 805 (1994)]. PCR was performed using PolB-5A and PolB-5B Final primers containing 1 μM of primer, 1 × PCR buffer and 60 μM of all four dNTPs Performed in a reaction volume of 50 μl. The reaction mixture was heated at 95 C for 3 minutes. 2.5 units of Ta q Add polymerase to start amplification, 1 minute at 95 ° C, 1 minute at 60 ° C and 2 minutes at 72 ° C. The 35 minute cycle lasted 35 cycles.   To clone the PCR-amplified fragment, use linear primers with 1 μl of each PCR product. Ligation was performed on T7Blue T-vector (Novagen, Madison, WI). Using the binding product Transform competent JM109 cells and add 40 μg / ml X-Gal, 40 μg / ml IPTG and On an LB plate containing 50 μg / ml ampicillin and white A clone containing the 7Blue T-vector was selected. Each PCR sample (i.e., wild-type, For the variants 1 and 2), 5 independent colonies were picked and 50 μg / ml The cells were grown overnight in 2 ml of LB medium containing nicillin. The alkaline lye described in Example 32 Plasmid DNA was isolated using the Niprep protocol.   PolB-5A and PolB-5B primers were used to analyze the cloned fragment. μM, 1X PCR buffer, all 4 dNTPs at 60 μM and 2.5 units of Taq polymerizer Using 50 μl of the reaction containing the enzyme, 1 μl of plasmid DNA from each clone was Amplified by PCR. PCR cycle at 95 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 2 minutes For 35 cycles. PCR products were purified with 6% native acrylic acid in 0.5X TBE buffer. The clones that were separated by electrophoresis on a midgel and generated a 620 bp fragment were further separated. Selected for analysis.   To perform sequencing, plasmid DNA from selected clones was PEG purification was performed as described in 32. 2 μl (about 100 ng) of PEG purified plasmid DNA Used for cycle sequencing reactions using a kit (Promega, Madison, WI). Plastic For biotinylation of the immer, use the Oligo Nucleotide Biotin Labeling Kit (Amersham) It was performed using. The sequencing reaction was performed with 8% denatured acrylic as described in Example 19. Separated on amide gel, transferred to nylon membrane and visualized. Wild type, mutant 1 and The DNA sequences of the 620 bp rpoB gene fragment obtained from the two mutant strains are listed in SEQ ID NOs: 122 to 124. I can. The sequence of the sense strand of each of the three TB strains is shown in the top row of SEQ ID NOS: 122 to 124. Show. The sequence of the antisense strand for wild-type, mutant 1 and mutant 2 strains These are listed in SEQ ID NOs: 125 to 127, respectively. ii) Preparation of Mycobacterium tuberculosis rpoB gene substrate   CFLP^TMTo generate substrates for use in the reaction, wild-type, mutant 1 and mutant 2rp A 620 bp cloned fragment obtained from the oB gene was amplified using PCR. PCR is used to The PCR product generated by the PCR process is responsible for the sense and antisense strands of the rpoB gene fragment. One primer of the primer pair labeled at the 5 'end to allow for analysis of any Performed using a limer. To generate a labeled substrate on the antisense strand Next, 10 ng of plasmid DNA from the cloned sequence was transferred to 1 μM PolB-5A Primer (unlabeled) and Oligo Nucleotide Biotin Labeling Kit (Amersham) PolB-5B primer biotinylated at the 5 'end using IX PCR buffer -50 μl reaction containing 60 μM of all four dNTPs and 2.5 units of Taq polymerase Used as template in material. The reaction is performed at 95 ° C for 1 minute, at 60 ° C for 1 minute and at 72 ° C. The 2-minute cycle was repeated 35 times. Obtained biotin-labeled antisense strand The 620 bp PCR product containing was purified as described in Example 19. Purified fragments 20 μl of H_TwoDissolved in O.   In the sense strand of the 620 bp fragment of the rpoB gene fragment (wild type, mutant 1 and mutant 2) 5 'with TMR dye using ate Labeling Kit (Molecular Probes, Inc., Eugene.OR) PolB-5A primer with a labeled end and PolB-5B primer with no label PCR was performed using 1 μM each. The PCR reaction was performed in a 1X PCR buffer in a final volume of 100 μl. Buffer, 60 μM of all four dNTPs and 5 units of Taq polymerase and sequencing 10ng of plasmid DNA from defined clones as template It was taken. The reaction was cycled through 95 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 2 minutes. The part was repeated 35 cycles.   In addition to the above PCR conditions, perform a PCR reaction using dUTP instead of dTTP, and A contained PCR fragment was generated. Uridine-containing PCR fragments were analyzed in clinical laboratories It is a standard type of fragment. Changes due to a single base pair change within a 620 bp fragment CFLP from substrate^TMUridine-containing PCR fragments to generate patterns A 5 'TMR label on the sense strand and a thymidine Instead, an rpoB gene fragment containing uridine was generated as follows. 1.5mM Mg Cl_Two2.5mM MgCl instead of_TwoUsing four dNTPs (ie, dATP, dCTP, dGTP and dTTP). TP) 100 μM dATP, 100 μM dCTP, 1 Except for using 00 μM dGTP and 200 μM dUTP, the 5 ′ end of the sense strand was labeled with TMR. According to the PCR protocol described above for the generation of a fragment, the uridine-containing 620 bp fragment (Wild type, mutants 1 and 2) were amplified.   620 bp P including sense strand (containing uridine or thymidine) labeled with TMR The CR product was purified on a 6% denaturing gel as described above and as described in Example 19. Elute overnight, precipitate with ethanol, and add 20 μl to a concentration of approximately 15 fmoles / μl. H_TwoDissolved in O. iii) Cleavage reaction conditions   Open for analysis of 620 bp rpoB fragment containing biotin-labeled antisense strand The cleavage reaction conditions are as follows. 6 μl of biotin-labeled PCR product is added to 1 μl of 10 X CFLP^TMBuffer (100 mM MOPS, pH 7.5, 0.5% Tween 20 and NP-40, respectively) and 25ng Cleavase^TMCombined with BN enzyme. Prior to the initiation of the cleavage reaction, the DNA mixture Was denatured by incubating at 95 ° C. for 10 seconds. The reaction is then added to 60 ℃, 2mM MnCl_TwoThe reaction was started by adding 1 μl. The cleavage reaction is Performed at 60 ° C. for 2 minutes. The cleavage reaction is performed by adding 5 μl of stop buffer. Stopped two minutes later. Run 6 μl of each sample on a 6% denaturing polyacrylamide gel. Separated by electrophoresis and labeled fragments as described in Example 19. Visualized. The obtained autoradiogram is shown in FIG.   In FIG. 78, lanes marked with M are input from Amersham (Arlington Heights IL). Contains biotinylated molecular weight markers, 200, 300, 400, and 500 nucleotides in length Includes bands corresponding to the Marker and uncleaved rpoB substrate (620 The size in parentheses is indicated on the left side of the autoradiograph using large arrows. Lane 1 ~ 3 are for cleavage of antisense labeled mutant 1, wild type and mutant 2 substrates Respectively containing the reaction products generated from the reaction. Point mutation from 5 'end label ( Distance (relative to wild-type sequence) is 511 nucleotides per variant 1 substrate, There were 499 nucleotides for two substrates.   From the results shown in FIG. 78, from each of the rpoB substrates labeled on the antisense strand, A similar but clearly different cleavage pattern was shown to be generated. field Compared to the cleavage pattern generated by the native substrate, the cleavage of mutant 1 substrate Band A has disappeared in the generated pattern. Opening of wild type and mutant 2 substrates Comparing the patterns generated by the cleavage, the intensity of band B was found to be higher in the mutant 2 substrate. Significantly decreased. Thus, the two mutants are distinguished from each other from the wild type. Can be   Cleavage reaction conditions for analysis of the 620 bp rpoB fragment containing the TMR-labeled sense strand It is as follows. 4 μl of the TMR-labeled PCR product was cleaved as described above. Open The cleavage reaction was performed using 5 μl of 95% formamide, 10 mM EDTA and 0.02% methyl violet (S igma) was stopped.   The reaction was heated to 85 ° C for 2 minutes and 5 μl of each reaction mixture was added to a buffer containing 0.5X TBE. Electrophoresis on 12% denaturing polyacrylamide gel (7: 1 cross-link) containing 7M urea in fur To separate. The gel was run at 33 watts for 1.5 hours. Labeled anti The reaction products were visualized using an FMBIO-100 Image Analyzer (Hitachi). Got The fluoroimager scan is shown in FIG. Run gel for another hour After electrophoresis, the second scan is shown in panel B.   FIG. 79 shows two panels A and B. Panel B is as shown in Panel A 2 shows a scan of the same gel as shown in Panel A after a longer time electrophoresis. doing. Therefore, panel B has a band pattern seen in the upper part of panel A. (The line connecting panels A and B indicates the area of expansion.) There). In panels A and B of FIG. 79, lanes 1-4 are mutant 1, wild type, On the sense strand, obtained from the mixture of mutant 2 and wild-type and mutant 2 substrates, respectively. Includes reaction products generated by cleavage of thymidine-containing substrates with TMR-labels It is. Lanes 5 of Panels A and B are labeled with TET as a marker. It contains a 157 bp fragment obtained from exon 4 of the rosinase gene (SEQ ID NO: 27). Panels A and B, lanes 6-9 are mutant 1, wild type, mutant 2 and wild type. Uridines with TMR-label on the sense strand, each obtained from a mixture with the variant 2 substrate Contains the reaction products generated by cleaving the containing substrate. Wild type and mutation The mixture with body 2 substrate (lanes 4 and 9) is mixed with 5 μl of each substrate after the cleavage reaction. And then 6 μl of the mixture was added to the gel. From 5 'end label The distance of the point mutation (relative to the wild type sequence) is 100 nucleotides per mutant 1 substrate It was 116 nucleotides for the tide, mutant 2 substrate.   The results shown in FIG. 79 show similar results for each of the rpoB substrates labeled on the sense strand. And clearly different cleavage patterns are produced. Each panel The left set of files shows CFLP generated from PCR products containing dNTPs.^TMIncluding patterns , The set on the right shows CFLP generated from PCR products in which dUTP was replaced by dTTP.^TMPatter Including CFLP generated from mutant 1 and wild-type dNTP-containing amplicon^TMPa When the turns are compared, the sequence changes around this mutant (100 bp from the labeled 5 'end) ), The intensity of the band (band A) of about 80 nt from the labeled 5 ′ end is significantly reduced. It turns out that it is less. In addition, vans that migrate about 200 to 250 nt from the labeled 5 'end (Band B) disappears in mutant 1. On the other hand, from wild type and mutant 2 Comparing the generated patterns, the disappearance of the band (Band C) of about 120 nt from the 5 'end Became clear. Further examination of the gel region corresponding to 120 nt revealed that panel B Shows that in mutant 2, band D is shifted downward with respect to the wild type. Was done. In panel B, another band migrating just above band D (labeled band D) Seems to shift downward with respect to the wild type in mutant 2. Review each panel Lane 4 mixed aliquots from wild-type and mutant CFLP reactions before electrophoresis However, this shift in mutant 2 (in band D) is actually present Is not due to electrophoresis artifacts .   CFLP generated from dUTP-containing amplicon^TMAfter examining the patterns, The ability to distinguish mutants from each other, as well as from wild-type, was achieved by replacing dUTP with dTTP It was shown that it was not adversely affected and could actually be enhanced. In this embodiment, the pattern When mutants were generated from amplicons containing dUTP but not dTTP, And both are more easily distinguished from the wild type. In the right part of panel A Comparing the lanes containing mutant 1 and wild type, a clear distinction between the two amplicons Some differences are evident, others are new and anticipated. I can not do such a thing. Specifically, band A is the same as in the left part of this panel. Thus, the intensity in the mutant was reduced compared to the wild type. A buffer that migrates at about 110 nt Band (band E) was found to be missing from the mutant, as did the band at about 250 nt. (Compare band B on the left side of the gel). Stronger labeled bands F indicates that there is no significant difference between the three samples containing dTTP, but the dUTP-containing amplifier Much stronger in wild-type patterns generated from recon than in mutants No. Comparison of the patterns generated from wild-type and mutant 2 shows that in some cases The differences were also revealed. The most notable are the two that move to about 150nt Like the new band complex (band H), the band that moves about 60 nt is the mutant 2 (Band G). Interestingly, that of these patterns Some of the factors that make each distinguishable from the others are dTT in PCR amplification. Most of the cleaved fragments are identical in the two experiments, except that P is replaced by dUTP. This result may be due to the change in secondary structure stability or the modification of dUTP by substitution of dUTP. Cleavase for secondary structures containing nucleotides^TMThe result of a change in the affinity of the enzyme This suggests that only minor changes occur in the resulting single-stranded DNA substrate. dUTP included Cleavase for secondary structure of DNA fragment^TMThese differences when recognized by Provides unexpected benefits of using such nucleotide substitutions Things. B) CFLP of a mutation in the KatG gene of M. tuberculosis^TManalysis i) Preparation of plasmid containing KatG gene sequence   KatG inheritance associated with M. tuberculosis or isoniazid resistance Genomic DNA isolated from M. tuberculosis strains containing mutations in offspring J. Obtained from Dr. Uhl (Mayo Clinic, Rochester, MN). These four strains Are referred to as wild-type, S315T, R463L and S315T; R463L [Cockerill, III et al. In fect. Dis. 171: 240 (1995)]. Strain S315T contains a G at codon 315 of the wild-type KatG gene. → Includes C mutation. Strain R463L has a G → T spike at codon 463 of the wild-type gene. The strain S315T; R463L contains a G → C mutation at codon 315 and codon 4 Includes both 63 G → T mutations.   The 620 bp region of the Mycobacterium tuberculosis KatG gene was subjected to PCR from DNAs derived from the above four strains. Amplified using Primers used to amplify the KatG gene sequence KatG904 [5'-AGCTCGTATGGCACCGGAAC-3 '(SEQ ID NO: 128)] and KatG1523 [5 '-TTGACCTCCCACCCGACTTG-3' (SEQ ID NO: 129)]. These primers Amplifies the 620 bp region of the KatG gene spanning both the S315T and R463L mutations I do. PCR was performed with 0.5 μM KatG904 and KatG1523 primers, 1 × PCR buffer And a final reaction volume of 100 μl containing 60 μM of all four dNTPs. Reaction mixing Heat the product at 95 ° C for 3 minutes, then amplify by adding 5 units of Taq polymerase And continue 35 cycles of 95 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 2 minutes. Was.   To clone the PCR amplified KatG fragment, 1 μl of each PCR product was Used for ligation to pT7 blue T-vector (Novagen, Madison, WI) did. Transforming competent JM109 cells using the ligation product, A clone containing the pT7 blue T-vector with the insert was cloned into 40 μg / ml X-Gal, 40 White on LB plate containing μg / ml IPTG and 50 μg / ml ampicillin Selected by. For each of the four PCR samples, four colonies were They were picked and grown overnight in 2 ml LB medium containing 50 μg / ml carbenicillin. Plasmid DNA was prepared using the alkaline miniprep protocol described in Example 32. Isolated using a col.   To analyze the cloned KatG fragments, 1 μl of plasmid from each clone was used. DNA with 0.5 μM KatG 904 and KatG 1523 primers, 1 × PCR buffer, 100 μl containing 60 μM of all four dNTPs and 5 units of Taq polymerase Was amplified by PCR using the above reaction solution. PCR was performed at 95 ° C for 1 minute and at 60 ° C 35 cycles of 1 minute and 2 minutes at 72 ° C. Reduce PCR product by 0.5X TBE Separated by electrophoresis on a 6% natural polyacrylamide gel in the buffer, 620 bp Clones producing fragments were selected for further analysis.   For sequencing purposes, plasmid DNA from selected clones was PEG purification was performed according to the protocol described in Example 32. 2 μl of plasmid DN A (about 100 ng) using KatG 904 and KatG15 containing a biotin moiety at the 5 'end. Fmol with 23 primers^RCycle using kits (Promega, Madison, WI) Sequencing was performed. Biotinylation of primers is performed using oligonucleotide This was performed using an otin labeling kit (Amersham). The sequencing reaction is Separated on an 8% denaturing polyacrylamide gel, transferred to a nylon membrane, and Visualization was as described in Example 19. Wild-type and mutant strains S315T, R46 3L and S315T; the DNA sequence of the 620 bp katG gene fragment from R463L, The numbers 130 to 133 are shown. The sense strand for each of the four katG gene fragments The sequences are shown as the top row of SEQ ID NOs: 130-133, respectively. Wild type and protruding Of the 620 bp katG gene fragment from the mutant strains S315T, R463L and S315T; R463L. The sequences of the chisense strand are shown in SEQ ID NOs: 134 to 137, respectively. ii) Preparation of M. tuberculosis katG gene substrate   CFLP^TMWild-type as well as S315 to make substrates for use in the reaction Cloning of T, R463L and S315T; R463L from M. tuberculosis strain 62 The 0 bp fragment was amplified using PCR. PCR was performed with 0.5 μM each of KatG 904 and K atG 1523 primer, 1X PCR buffer, 60 mM, all 4 dNTPs, 5 units Plus Taq polymerase as a template from sequenced clones Performed in a final reaction volume of 100 μl containing 10 ng of mid DNA. Anti The reaction was performed at 95 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 2 minutes for 35 cycles.   To obtain a 620 bp PCR fragment of the KatG gene with a biotin label on the sense strand In addition, an unlabeled KatG 1523 primer (SEQ ID NO: 129) and a 5'-biotinylated Kat G904 primer (SEQ ID NO: 128) was used in the PCR. Biotinylation This was performed using a gonucleotide biotin labeling kit (Amersham). Antisen For the production of a similar fragment having a TMR label on its strand, an unlabeled KatG 904 (SEQ ID NO: 128) and TMR-labeled KatG 1523 (SEQ ID NO: 129) primer Used in. The amplified PCR product was purified on a 6% denaturing gel, eluted overnight, Precipitate with ethanol and add 50 μl H as described in Example 19._TwoRedissolved in O. iii) Cleavage reaction conditions   The cleavage reaction conditions for analyzing the KatG substrate labeled on the sense strand are as follows. It was. 5 μl of the biotin-labeled PCR product was added to 1 μl of 10 × CFLP^TMBuffer (100 mM MOPS, pH 7.5, 0.5% each of Tween 20 and NP-40) and 25 ng Cleavase^TMCombined with BN enzyme. Before starting the cleavage reaction, mix the DNA The mixture was denatured by incubating at 95 ° C for 10 seconds. Then the reaction The mass was cooled to 50 ° C. and 1 μl of 2 mM MnCl_TwoThe reaction was started by adding . The cleavage reaction is incubated at 50 ° C for 2 minutes and 5 μl of stop buffer is added. And stopped by. Transfer 4.5 μl of each sample to 10% denaturing polyacrylamide gel. After transfer to a nylon membrane as described in Example 19, the labeled fragments were visualized. . The obtained autoradiogram is shown in FIG.   In FIG. 80, the lane marked "M" is Amersham (Arlington Heights, IL) 50, 100, 200, 300, 400, 500, including biotinylated molecular weight markers obtained from , 700 and 1000 nucleotides in length. Marker size This is indicated using a large arrowhead. Lanes 1-4 are Cleavase^TM In the presence of BN enzyme, R463L, R463L, respectively, S315T, S315T and wild-type KatG substrate Includes reaction products obtained by incubation. 5 'end of mutation Distance from the sense is the R463L mutation if the label is on the sense strand. Is 485 nucleotides for the S315T mutation and 41 nucleotides for the S315T mutation. is there.   The results shown in FIG. 80 show that the wild-type and S315L mutant (S 315T and S315T; found on both R463L substrates) But a distinctly different cleavage pattern was generated. Wild-type fragment (lane CFLP for 4)^TMCompared to the pattern, the S315T mutation (R463L; S315T and And S315T mutants (found in lanes 2 and 3)) Band B located about 40 nucleotides from the end label of the raw substrate disappears You can see that The disappearance of band B was determined by the distance of the S315T mutation from the 5 'end (the (41 nucleotides from the 5 'end label on the sense strand). Subsequent experiments Causes the R463L mutant to move about 500 nt from the 5 'end label on the sense strand. (A downward band shift in the R463L mutant) Indicates that it can be distinguished from wild type, but it is difficult to develop with many gel systems. You.   The cleavage reaction conditions for analyzing the KatG substrate labeled on the antisense strand As described for the strand. 4.5 μl of each sample is 10% denatured polyacrylic acid. The gel was run on a ruamide gel and the labeled fragment was analyzed for H 2 as described in Example 33 (a) (iii). Visualization was performed using an itachi FMBIO-100 fluorescence image analyzer. Fig. 81 shows the obtained scan. Show.   In FIG. 81, the lane marked with “M” was digested with MspI and described in Example 8. The 3 'end using terminal deoxynucleotidyl transferase as described above. Contains plasmid pUC19 DNA labeled with fluorescein ddUTP. This marker The bars correspond to lengths of 110/111, 147, 190, 242, 331, 404, 489 and 501 bp. Incl. Additional 26, 34, and 67 bp marker bands are apparent in this figure. Or not. Marker size is indicated using a large arrowhead. Lanes 1-4 are Cleavase^TMIn the presence of the BN enzyme, R463L, S315T, respectively; R463L, S315 Includes reaction products obtained by incubating T and wild-type KatG substrates. No. The position of the single base mutation from the 5 'end label is labeled on the antisense strand. If present, 136 nucleotides for R463L mutation And 580 nucleotides for the S315T mutation.   The results shown in FIG. 81 indicate that the wild type mutant has an R463L substitution on the antisense strand. It can be distinguished from S315T; R463L double mutant or R463L mutant When compared to the lane containing alone, the R463L mutation shows a strong band migrating at about 130 nt. It can be seen that this is related to the existence of (band A). This result is combined with the result shown in FIG. Taken together, all three of these mutants are CFLP^TMAnalysis distinguishes each other It can be seen that it can be distinguished from the wild type as well.   CFLP^TMTechnology reduces gel electrophoresis processing time from 12-18 hours to 5-10 minutes This is advantageous in terms of cost. Readout compatible with multi-lane fluorescence image detector Allows simultaneous processing of up to 48 reactions, thereby increasing the processing volume It is. As a result, the time required to perform the analysis is reduced, making it necessary to report results to patients. Time is reduced from days to hours, or for MDR-TB patients, Reduced from weeks to hours. Early detection of MDR-TB has been Reduced costs of long stays in isolated wards while investigating the efficacy of treatment Can save thousands of dollars per person.                                 Example 34                  Rapid identification of bacterial strains by CFLP ^™ analysis   The results shown above indicate that CFLP^TMAnalysis can be used to determine the genetics associated with drug resistance By examining some of the offspring (eg, rpoB and katG), M. tuberculo sis) wild type and the presence of drug-resistant mutations Was. CFLP^TMWhether the assay can be used as a detection and identification method for various microorganisms CFLP to find out^TMAnalysis derived from bacterial 16S rRNA gene Performed using the substrate.   The bacterial 16S rRNA gene varies throughout the phylogenetic tree. These genes Contains segments that are conserved at the species, genus or kingdom level. These features Using the signature, a primer containing a consensus sequence flanking the variable region was created . These primers are used to augment a segment of the bacterial 16S rRNA gene. Width. The segment was then used for Southern blot hybridization [G reisen et al. Clin. Microbiol. 32: 335 (1994)] or SSCP analysis [Widjojoa tmondjo et al. Clin. Microbiol. 32: 3002 (1994)]. Analysis of these types is faster than traditional culture methods, but at most, specific genera and higher It only distinguishes species within a bacterial taxon. However, often different fungi of the same species It is desirable to distinguish between strains. For example, if a given species is a harmless organism and disease May contain subspecies, including protozoa. Possible to distinguish between species and / or subspecies CFLP to develop technology^TMAnalysis of bacterial 16S rRNA gene Applied to derived segments. A) Bacterial strain   Table 3 below lists the bacterial strains used in this study. These strains are Deutsc he data derived from a deposit obtained from Sammlung von Mikroorganismen (DSM). Desulfurococcus amylolyticus strain Z-53 With the exception of 3, they were derived from the ATCC strains listed below.   The strains listed in Table 3 include E. coli strains B and K-12 and desulfur Except for Coccus amylolyticus (Desulfurococcus amylolyticus), Represents a microorganism. Desulfurococcus amylo lyticus) uses common primers based on rRNA gene sequences whose design is known. Using rRNA fragments from archaeal species whose rRNA gene sequence has not been reported. The gene fragment sequence was included in this study to see if it could be amplified. table The strains listed in No. 3 can be from several different genera (eg, Escherichia). , Shigella, Salmonella and Campylobacter bacter)) to provide representative examples, as well as different species (and Was selected to provide some representative examples of subspecies). For example, three types of large Enterobacteriaceae (E. coli) strains contain CFLP produced by cleavage of rRNA gene substrates.^TMBa Pattern matching (or lack thereof) can be examined between species within a given genus. I chose. In addition, E. coli serotype O157: H7 indicates that this strain is hemorrhagic. The relationship was deep in the development of colitis and was examined. E. coli strain B and CFLP observed by cleavage of rRNA gene substrate derived from K12 and K-12^TMPattern is Produced by cleavage of rRNA gene substrate from E. coli serotype O157: H7 It was interesting to find out if it was different.   Table 4 below shows the phylogenetic relationships between the strains used in this example. B) Microbial growth   To minimize the treatment of pathogenic strains, the growth of microorganisms should be The culture was performed by surface culture or plate culture. i) Escherichia, Shigella, Salmonella And Staphylococcus spp.   All strains were obtained from the ATCC strains listed in Table 3 above as follows. Trip Ticase soy broth and 15% glycerol (Remel Corp., Lenexa, KS. Log the culture of one platinum loop, previously frozen in Log 06-5024), onto trypticase soy agar. It was subcultured on a slope (Remel. Catalog 06-4860). Incubate culture at 37 ° C overnight Was added. ii) Propagation of Campylobacter species   Trypticase soy broth and 15% glycerol (Remel Corp., Lenexa, KS. One loop of the culture previously frozen in catalog 06-5024) was used for 10% sheep blood, Hotelricin B, cephalotin, trimethoprim, vancomycin and polymiki Subculture on Campylobacter agar supplemented with Syn-B (BBL, catalog 21727) Was. Place the inoculated plate in a Campy microaerobic pouch (BBL, catalog 4360656) Closed and incubated at 42 ° C for 3 days. C) Extraction of genomic DNA from microorganism   For each bacterial sample, 300 μl TE buffer and 300 μl phenol: Loroform: Isoamyl alcohol (25: 24: 1) in a 1.5 ml microcentrifuge tube I put it. This mixture is called the extraction buffer. 1 loop (about 0.1 ml) of the desired bacterial strain Remove from slant culture or plate culture and extract in 1.5 ml microcentrifuge tube Combine with buffer and vortex the contents for 2 minutes. DN extracted in aqueous phase A was processed for further purification as described below.   E. coli and C. Ethanol precipitate the sample of the jejuni strain and dissolved in 1 TE buffer. The sample was then incubated at 37 ° C. for 30 minutes with 0.5 μg RN Treated with A. The DNA was ethanol precipitated, collected by centrifugation, and It was dissolved in 0 μl of 10 mM Tris-HCl (pH 8.0, 25 ° C.).   Shigella, Salmonella and Staphylococcus (Staph ylococcus)^TM50 μl using 30 filters (Amicon) Concentrate to Microspin^TMUsing an S-200HR gel filtration column (Pharmacia Biotech) Transferred to TE buffer. The sample was then incubated at 37 ° C. for 80 minutes with 0.5 μg of RNase A. Processed. The DNA was ethanol precipitated, collected by centrifugation, and 200 μl It was dissolved in 10 mM Tris-HCl (pH 8.0, 25 ° C.).   The genomic DNA of E. coli strain B (ATCC 11303) was ligated to Pharmacia Biotech (Pi scataway, NJ; catalog 27-4566-01, lot 411456601). DNA Was dissolved in 10 mM Tris-HCl (pH 8.0, 25 ° C.).   Desulfurococcus amylolyticus strain Isolate genomic DNA from Z-533 (DSM3822) and use standard techniques for cesium chloride centrifugation. Purified using techniques [Bonch-Osmolovskaya et al., Microbiology (Engl. Transl. O. f Mikrobiologiya) 57:78 (1988)].   The concentration of the genomic DNA preparation is determined by the OD of the preparation.₂₆₀Determined by measuring . D) Design of primers for amplification of 16s rRNA gene of bacterial species   Primes that allow amplification or detection of 16S rRNA sequences from various bacterial strains Markers and probes have been reported. These oligonucleotide primers Alternatively, probes may be obtained by comparing 16s rRNA gene sequences from different eubacteria species. Represents the consensus sequence obtained. For example, PCR amplification of bacterial rRNA gene sequences or Is an oligonucleotide primer suitable for dot blot hybridization [Eg, PCT Publication No. 90/15157; Widjojoatmodjo et al., J. . Clin. Microbiol. 32: 3002 (1994)]. Typically, the conserved sequence of the primer is Design the non-conserved region of the 16s rRNA gene having a species-specific sequence to be adjacent You.   A number of previously published common primers obtained from the 16S rRNA gene sequence , CFLP^TMThe substrate production ability for use in the reaction was examined. Primer 1638 , 1659 and 1743 are described in PCT Publication No. 90/15157. Primer ER10 is described in Widjojoatmodjo et al. (Supra). Primer SB-1, SB-3 And SB-4 represent new primers (ie, not previously published). The primers used in this example are listed in Table 5 below.   Oligonucleotide primers were purchased from Integrated DNA Technologies, Inc. From obtained. Oligonucleotides were added to 10 mM Tris-HCl (pH 8, 25 ° C.) at 20 μM. Dissolved in concentration. Two sets of primers were synthesized. One set has an OH group at the 5 'end (Ie, unlabeled primer), the other set has a fluorescent dye TET at the 5 'end. (A tetrachlorinated analog of 6-carboxyfluorescein, Applied Biosystems) (Ie, TET-labeled primers). TET-labeled primers Indicated by using "TET" as a subscript in the name of the immer (for example, TET- 1638 indicates a 1638 primer having a 5 'TET label. ).   The positions of each of the primers listed in Table 5 are shown in FIG. 82 in the E. coli rrsE gene. The sequence is shown along with the offspring (encoding 16S rRNA). In FIG. 82, the primer The columns are shown in bold and underlined are the primer sequence and the E. coli rrsE gene sequence. Used to indicate a perfect match between The sequence of the E. coli rrsE gene is , SEQ ID NO: 145. As shown in FIG. 82, the 1638, ER10, SB-1, SB-3, and SB-4 Primers correspond to sequences present on the sense strand of the 16S rRNA gene. 1659 , 1743 primer is used for the sequence present on the antisense strand of the 16S rRNA gene. Corresponding.   Figure 83 shows the E. coli rrsE gene (SEQ ID NO: 145), Cam. jejun5 remains Gene (rRNA gene from C. jejuni) (SEQ ID NO: 146) and Stp. aureus Provides alignment of the gene (rRNA gene from S. aureus) (SEQ ID NO: 147) You. 1638, ER10, 1659 (shown as the complement of 1659), SB-1, SB-3, SB-4 and 17 The position of the 43 (shown as the complement of 1743) primer is shown in bold. rRNA gene A gap (dash) is introduced to maximize alignment between.   In prokaryotes, 2-10 copies of the ribosomal RNA gene are present, and Escherichia ( Escherichia) averages 7 copies. By PCR amplification, these genes Are produced, essentially "multiplex" PCR from that strain. C FLP is a complex pattern of rRNA genes slightly altered in the microorganism. , No specific rRNA is directly involved in the entire “barcode”. How many In these cases, these small changes (between rRNA genes, for example, Enterobacteriaceae (see small changes between E. coli rRNA genes)^TMpattern Causes a small (small signal) band shift at, very closely related To enable the distinction between isolated organisms. Most or all copies of these genes Larger changes in the sequence can be seen when comparing less related organisms. (Eg, E. coli, C. jejuni and S. aureus in FIG. 83). (See widespread changes between rRNA genes), these large differences are In the turn, it is shown as a larger pattern change. Variability of these genes Nevertheless, amplification by PCR must be performed between conserved regions of the rRNA gene. All rRNA sequence collections for any microorganism of interest. You do not need to know in advance.   Using three primers (TET-1638, TET-ER10 and TET-SB-4), 16S A fragment was prepared in which the 5 'end of the sense strand of the rRNA gene was fluorescently labeled. The other two Two primers (TET-1659 and TET-1743) were used to create a labeled fragment of the antisense strand. It was used for production.   Using the indicated primer pairs, the 16S rRNA gene sequences from various bacterial The expected sizes of the PCR products produced by the amplification are shown in Table 6. Table 6 Thus, the expected size of the PCR product depends on the known distribution of the 16S rRNA gene of the indicated species. Based on column. In Table 6, the following abbreviations are used: That is, Dco (Desulfurococcu s), E.C. co (E. coli), Cam (Campylobacter) and Stp (Staphylococus). You. Position of the PCR product relative to the sequence of the E. coli rrsE gene (see FIG. 82) Locations are shown in the last column. E) PCR amplification of 16S rRNA gene sequence   For each primer pair listed in Table 6, 16S r from each bacterial strain listed in Table 3 The ability to amplify RNA gene sequences was examined. Commercialization of recombinant Taq DNA polymerase Preparations containing various amounts of the E. coli 16S rRNA gene sequence. Is knowledge. During amplification of a bacterial DNA sample, contaminating E. coli 16S To minimize rRNA sequence amplification, use AmpliTaq DNA polymerase, LD (low DNA) (Perkin Elmer) was used for PCR. This Taq DNA polymerase The product is tested by the manufacturer and placed in a standard 2.5 unit enzyme aliquot. Confirmed that less than 10 copies of bacterial 16S ribosomal RNA gene sequence were present Have been.   Each primer pair (Table 6) was tested in a PCR. PCR reaction solution is 10 mM Tris-HCl (pH 8.3, 25 ° C.), 50 mM KCl, 1.5 mM MgCl_Two, 0.001 % w / v gelatin, 60 μM each of dGTP, dATP, dTTP and dCTP, 1 each μM of one 5′-TET-labeled primer and one unlabeled primer, 2.5 units Knit AmpliTaq DNA polymerase, LD included. Reaction was performed by AmpliTaq D NA polymerase, LD. In a final volume of 50 μl using lot E0332 or Ampl iTaq DNA polymerase, LD. Run in a final volume of 100 μl using lot D0008 Was. The amount of genomic DNA added was 6-900 ng for each PCR. Bacteria Control reactions without the addition of nome DNA were also performed to generate AmpliTaq DNA polymerase. And the amount of 16S rRNA product produced by contaminants in the LD preparation.   PCR reaction is PTC-100^TMWith a programmed heat controller (MJ Research, Inc.) went. Two sets of cycle conditions were used. The first set of conditions is 95 ° C for 30 seconds, 60 ° C 30 cycles of 1 minute at 72 ° C. and 30 seconds at 72 ° C. Cooled to 4 ° C. The second set of conditions consisted of 95 ° C for 30 seconds, 60 ° C for 1 minute and 72 ° C for 90 minutes. The seconds were 30 cycles and after the last cycle, the tubes were cooled to 4 ° C. Sand The difference between the two cycle conditions is the time during which the reaction solution is held at the elongation temperature (72 ° C). You. These two extension times were tested for the expected size of the 16S rRNA target. However, depending on the primer pair used for amplification, 208-1388 bp Because it changes within the range.   As a rule of thumb, if the length of the target to be amplified is less than 500 bp, If using an extension step and the target length is 500-1000 bp, an extension step of 30-60 seconds If the target length is greater than 1 kb, extend approximately 1 minute per 1 kb length. Do. The first set of PCR conditions (30 second extension step) was performed with longer amplicons (amps). licons), but the yield was determined using a second set of PCR conditions (90 second extension). It was lower than when used.   After thermal cycling, 400 μl of formamide containing 1 mM EDTA was added to each sample. Added and the sample was concentrated on a Microcon 30 to a volume of 40 μl. Sample (40 μl) denaturing 6% polyacrylamide gel (7M urea, 0.5X TBE running buffer) The sample was loaded after pre-warming to 50-55 ° C. . Samples can be run at 20 W for 20 minutes (200-350 bp fragment) or 40 minutes (cut longer than 1 kb). Piece) electrophoresed. Gels were prepared using Fluorescent Me using 585 or 505 nm filters. Scan using thod Bio Image Analyzer Model 100 (FMBIO-100, Hitachi) Was.   From the results of these PCRs, each primer pair tested (Table 6) showed the expected large It was found that the size fragment was successfully amplified. That is, the primers shown in Table 6 Pairs include archaea, gram-positive and gram-negative bacteria, different species of the same genus and the same End-labeling D using genomic DNA from various prokaryotes such as different strains Suitable for NA fragment amplification. These PCRs are also based on the genomic DNA present in the PCR. Although the amount of NA varies from strain to strain, the yield of amplification product is always In AmpliTaq DNA polymerase, LD found in the reaction solution without the addition of Multiple times higher than trace amounts of product from E. coli genomic DNA did. F) Preparation of 16S rRNA gene substrate   CFLP^TMLabeled PCR corresponding to bacterial 16S rRNA sequence for use in reactions The following primer pairs were used in the PCR to generate the product. 1. Using the SB-1 / TET-1743 pair, Desulfurococcus amylolyticus (Desu lfurococcus amylolyticus) (DSM 3822), E. coli strain K-12 (ATCC 14 948); aureus subsp. aureus (ATCC 33591) and S. aureus. aureus subsp. aureus ( An approximately 297 bp fragment was amplified from genomic DNA derived from ATCC 33592). The resulting PCR The product contains a 5 'TET-label on the antisense strand. 2. Using the TET-SB-4 / 1743 pair, E. coli strain B (ATCC 11303), colon E. coli strain K-12 (ATCC 14948), E. coli serotype O157: H7 (ATCC 4389) 5), Shigella dysenteriae serotype 2 (ATCC 29027) and Salmonella (Sa lmonella) choleraesuis subsp. choleraesuis serotype typhi (ATCC 6539) An approximately 208 bp fragment was amplified from the nome DNA. The obtained PCR product is placed on the sense strand. Includes 5 'TET-label. 3. Using the 1638 / TET-1659 pair, E. coli strain B (ATCC 11303), colon E. coli strain K-12 (ATCC 14948), E. coli serotype O157: H7 (ATCC 4389) 5), Shigella dysenteriae serotype 2 (ATCC 29027) and Salmonella (Sa lmonella) choleraesuis subsp. choleraesuis serotype typhi (ATCC 6539) An approximately 350 bp fragment was amplified from the nome DNA. The resulting PCR product is antisense Include a 5 'TET-label on the strand. 4. Using the TET-ER10 / 1743 pair, E. coli strain K-12 (ATCC 14948) and Campylobacter jejuni subsp. jejuni (ATCC 33291) An approximately 1292 bp fragment was amplified from genomic DNA. The resulting PCR product is on the sense strand Contains a 5 'TET-label. 5. Using the 1638 / TET-1659 pair, E. coli serotype O157: H7 (ATCC 43895). ), Salmonella choleraesuis subsp. choleraesuis serotype typhi (AT CC 6539); Shigella dysenteriae serotype 2 (ATCC 29027); aureus subsp. aureus (ATCC 33591), S.A. aureus subsp. aureus (ATCC 33592), S.A. aureu s subsp. aureus (ATCC 13565), S.A. hominis (ATCC 29885) and S.P. warneri (AT An approximately 350 bp fragment was amplified from genomic DNA derived from CC 17917).   PCR was performed as described in E) above. Two separate PCR reactions are performed Campylobacter jejuni subsp. jejuni (ATCC 33291) Performed using 0.2 μg of nome DNA and the TET-ER10 / 1743 primer pair. One Reactions are performed in a final volume of 50 μl and are extended at 72 ° C for 30 seconds during thermal cycling. Used. The second reaction was performed in a final volume of 100 μl and extended at 72 ° C. for 90 seconds. It was used. The yield of the PCR product produced in the second reaction was 76% (the first reaction Higher than that). After the amplification reaction, as described in E) above, The pull was processed and electrophoresed on a denaturing polyacrylamide gel. Electrophoresis After, the desired band is excised from the gel and the gel section is excised with 0.5 M ammonium acetate, Elute by placing in 0.4 ml of a solution containing 0.1 mM EDTA and 0.1% SDS Was. The mixture was then incubated at 55 ° C for 2 hours and then at 37 ° C for 12 hours. The sample is concentrated to 25 μl using a Microcon 30 (Amicon) and Transferred into water using a pin column (Pharmacia). G) Cleavage reaction conditions   The cleavage reaction is performed with about 0.2-1 pmol of 5 'TET-labeled DNA group (as shown below). Quality, 10ng Cleavase^TMBN enzyme (Third Wave Technologies), 1X CFLP buffer and 0.2 mM MnCl_TwoPerformed in a final volume of 10 μl containing reaction The liquid is initially MnCl_TwoWas prepared as a 9 μl mixture containing no. This mixture Heat to 95 ° C for 10 seconds, then the desired incubation temperature (45 ° C, 50 ° C or 65 ° C). The optimal reaction temperature for each substrate is determined by the Based on the presence of some undigested substances to display molecules in a wide range up to Selected. The optimal temperature selected for each substrate is described in the discussion of FIGS. 84-87 below. Shown.   The cleavage reaction was performed with 1 μl of 2 mM MnCl_TwoWas started by adding. Desired temperature After 2 minutes incubation at 95 ° C, 95% formamide, 5 mM EDTA, 5% By the addition of 10 μl of a solution containing lyserol and 0.02% methyl violet The reaction was stopped. Uncleaved, or “no enzyme” controls are Cleavase^TMH instead of BN enzyme_TwoSame as above except that O was used In the same manner for each substrate. Transfer the sample (approximately 4-8 μl) to 45 mM Tris 6 to 1 with 7 M urea in buffer containing borate, pH 8.3, 1.4 mM EDTA Swim on 2% denaturing polyacrylamide gel (19: 1 cross-linking) at 15-20W for 9 minutes (Specific gel percentages are shown in the description of FIGS. 84-87 below). Then The gel was run on a FMB10-100 filter using a 585 nm filter. (Hitachi).   The resulting fluorescence image scans are shown in FIGS. Figure 84 shows the SB-1 / TET-1743 pair Desulfurococcus amylolyticus (DSM  3822), E. coli strain K-12 (ATCC 14948), S. aureus subsp. aureus (ATC C 33591) and S.I. aureus subsp. Genome DN derived from aureus (ATCC 33592) A cleavage product generated by cleavage of an approximately 297 bp 16S rRNA substrate generated using A Is shown. Lanes 1 to 4 are Cleavase, respectively.^TMIn the absence of BN enzyme Desulfurococcus amylolyticus (D SM 3822), E. coli strain K-12 (ATCC 14948), S. aureus subsp. aureus (A TCC 33591) and S.I. aureus subsp. aureus (ATCC 33592) Includes products generated by cuvating. Lanes 5-8 are cleaved, respectively. Alease^TMDesulfurococcus amylolyticus in the presence of BN enzyme (Desulfurococcus amylolyticus) (DSM 3822), E. coli strain K-12 (ATCC 1 4948); aureus subsp. aureus (ATCC 33591) and S. aureus. aureus subsp. aureus (ATCC 33592) by incubating the substrate Including. CFLP^TMThe reaction was performed using about 1 pmol of each PCR product, and the cleavage reaction Was incubated at 50 ° C. for 2 minutes. The cleavage product is, as described above, 8% It was developed by electrophoresis on a acrylamide gel.   The results shown in FIG. 84 indicate that Desulfurococcus amiloriticus (Desulfurococcus  amylolyticus) (DSM 3822), E. coli strains K-12 (ATCC 14948) and S. au reus subsp. Clear CFLP with the use of aureus substrate^TMThe pattern can be obtained And Two types of S. aureus subsp. Cleavage of aureus substrate (lanes 7 and 8) By the same CFLP^TMA pattern occurred. These two types of S. aureus subsp. aur eus strains (ATCC 33591 and 33592) contain the antibiotics methicillin and gentamicin. Are considered to be different subspecies based on differences in their susceptibility to infection. this Resistance or susceptibility to these antibiotics indicates that mutations in the 16S rRNA gene Not relevant. Thus, the use of certain 16S rRNA substrates may result in different CFLP^TMPa The turn was not expected to be confirmed.   The results shown in FIG. 84 indicate that Desulfurococcus a Desulfurococcus amylolyticus (DSM 3822), Escherichia coli (E. coli) Strain K-12 (ATCC 14948) and S. aureus subsp. Aureus identification and differentiation possible CFLP^TMIt shows that a substrate for analysis can be generated.   In FIG. 85, panel A shows the TET-SB-4 / 1743 pair and E. coli strain B (ATCC 11303), E. coli strain K-12 (ATCC 14948), E. coli blood Clear type O157: H7 (ATCC 43895), Shigella dysenteriae serotype 2 (ATCC 2 9027) and Salmonella choleraesuis subsp. choleraesuis serotype Approximately 208 bp 16SrRN generated using genomic DNA from typhi (ATCC 6539) A shows the reaction product generated by cleavage of the A substrate. TET-SB-4 / 1743 pair Amplify a part of the 16S rRNA gene located in the 3 'region (see Fig. 82).   85, CFLP shown in Panel A^TMThe reaction uses about 0.7 pmol of each PCR product. The cleavage reaction was incubated at 50 ° C. for 2 minutes. The cleavage products are shown in FIG. By electrophoresis on an 8% denaturing polyacrylamide gel as described above. Expanded.   In FIG. 85, panel B shows the 1638 / TET-1659 pair and E. coli strain B (ATCC 11303), E. coli strain K-12 (ATCC 14948), E. coli serum Type O157: H7 (ATCC 43895), Shigella dysenteriae serotype 2 (ATCC 290 27) and Salmonella choleraesuis subsp. choleraesuis serotype ty Approximately 350 bp 16S rRNA group prepared using genomic DNA derived from phi (ATCC 6539) 2 shows the reaction products generated by cleavage of the quality. The 1638 / TET-1659 pair is 5 'of the gene Amplify a part of the 16S rRNA gene located in the region (see FIG. 82).   85, CFLP shown in Panel B^TMThe reaction is performed using approximately 1 pmol of each PCR product. The cleavage reaction was incubated at 45 ° C. The cleavage product is 8% polyacrylic acid. It was developed by electrophoresis on a luamide gel.   The lanes marked “M” in panels A and B of FIG. 85 are the plasmids digested with MspI. PUC19 DNA and terminal deoxynucleotides as described in Example 8. 3 'labeled with fluorescein ddUTP using thiotransferase Including the end. The markers are 26, 34, 67, 110/111, 147, 190, 242 and 331b Includes bands corresponding to length p. Another marker band at 404, 489 and 501 bp is It is not clear in this figure. In panel A, lanes 1-5 are cut off Lanes 6-10 each contain no (i.e., no enzyme) controls, E. coli strain B (ATCC 11303), E. coli strain K-12 (ATCC 14948), Enterobacteriaceae (E. coli) serotype O157: H7 (ATCC 43895), Shigella dysenteriae Serotype 2 (ATCC 29027) and Salmonella choleraesuis subsp. ch By incubating the substrate from oleraesuis serotype typhi (ATCC 6539) Includes the resulting cleavage products. In panel B, lanes 1-5 are not cut Lanes 6-10 each contain a control (i.e., no enzyme) and each of E. coli (E. . coli K. (ATCC 14948), E. coli strain B (ATCC 11303), E. coli (E. coli) serotype O157: H7 (ATCC 43895), Shigella dysenteriae serum Type 2 (ATCC 29027) and Salmonella choleraesuis subsp. choler Produced by incubating substrate from aesuis serotype typhi (ATCC 6539) Cleaved cleavage products.   The lower molecular weight material found in the "uncleaved" lane is dH_TwoIn O After storage for several days. This decomposition EDTA is not present in the storage solution (i.e., trace amounts of required metal ions are present) May be due to surrounding nucleases that are active in such cases. This Degradation is effectively suppressed by including tRNA in the storage solution (Example 19). Seen in these uncut controls (panel B, lanes 1-5). Decomposition is CFLP^TMHas no effect on the result.   The results shown in Figure 85 show that some regions of the 16S rRNA gene are more variable than others. Analysis of these areas will compare very closely related organisms It is particularly useful in some cases. For example, 1638 / TET-1659 pair (gene Amplifies a portion of the 16S rRNA gene located in the 5 'region) Using quality, Escherichia, Shigella and Salmo Distinguish between DNAs from the genus Salmonella (panel B, lanes 6-10) In addition, the three E. coli strains tested (ie, strain B, K-12 And 0157: H7) (panel B, lanes 6-8) also clearly different from DNA CFLP resulting in cleavage pattern^TMPatterns can be generated.   In contrast, an approximately 208 bp fragment located near the 3 'end of the 16S rRNA gene In the case of a DNA fragment produced by using the resulting TET-SB-4 / 1743 pair, CFLP between strains of the Escherichia-Salmonella group^TMputter No substantial difference was observed (Panel A, lanes 6-10). 16S rR This contrast in the degree of variability between the 5 'and 3' regions of the NA gene Comparison between strains of the Lysia (Escherichia) -Salmonella group Consistent with results reported by Widjojoatmondjo et al. (Supra) doing.   Each organism has multiple copies of the 16S rRNA gene, which are Because of the co-amplification, different amplification products from the same organism can produce the same cleavage pattern. It was important to show. In FIG. 86, Campylobacter butter (Campylobacter) jej uni subsp. jejuni (ATCC 33291) from two separate PCR reactions, TET-E By cleavage of the approximately 1292 bp 16S rRNA substrate created by using the R10 / 1743 pair The cleavage products generated are shown in lanes 2 and 3. For comparison, E. coli The same region amplified from strain K-12 (ATCC 14948) is shown in lane 1. CFLP^TMreaction Was performed using about 60 fmoles of each PCR product, and the cleavage reaction solution was incubated at 50 ° C. for 2 minutes. Incubated. The reaction was stopped with 95% formamide, 5 mM EDTA, 5% Performed by the addition of lyserol and 0.02% methyl violet. Cleavage products Developed by electrophoresis on a 6% denaturing polyacrylamide gel as described above did.   The results shown in FIG. 86 show very different CFLP^TMThe pattern is purple bacteria (Purplebac teria) (Escherichia, lane 1) and ε (campylobacter (Campylobacter), lanes 2 and 3) produced using substrates from the subphylum But the same CFLP between the products of individual PCR reactions on the same genomic DNA^TMPatter In this case (lanes 2 and 3).   In FIG. 87, the 1638 / TET-1659 pair and E. coli serotype O157: H7 (ATCC 43895); choleraesuis subsp. choleraesuis serotype typhi (ATCC 6539), Shige Shigella dysenteriae serotype 2 (ATCC 29027); aureus subsp. aureus (ATCC 33591), S.M. aureus subsp. aureus (ATCC 33592), S. aureus subsp. aureu s (ATCC 13565), S.P. hominis (ATCC 29885) and S.P. from warneri (ATCC 17917) Generated by cleavage of an approximately 350 bp 16S rRNA substrate made using genomic DNA The resulting cleavage products are shown in lanes 1-8, respectively. CFLP^TMReaction is about 200 fmol Of each PCR product as described above, and the cleavage reaction was incubated at 65 ° C for 2 minutes. Incubated. The cleavage product is 10% denatured polyacrylamide as described above. It was developed by electrophoresis on Dogel.   The results shown in FIG. 87 show very different CFLP^TMThe pattern is red bacteria (lane 1) -3) and DNA derived from a strain representative of the Gram-positive phylum (lanes 4-8) Indicates that it was generated using. CFLP^TMThe real difference between the patterns is , Escherichia (lane 1), Salmonella (lane 2) ) And Shigella (lane 3).   In addition, CFLP^TMThe substantial difference between the patterns is that Staphylococcus aus Reus (Staphylococcus aureus) (lanes 4-6), hominis (lane 7) and warneri (lane 8) was detected among the species. Staphylococcus aureus ( Staphylococcus aureus) subsp. aureus ATCC 33591 (lane 4), ATCC 33592 (lane 4) Between the three strains, lane 5) and ATCC 13565 (lane 6), CFLP^TMPa No substantial differences were found in the turns. These S. aureus isolates reported RRNA genetics differ but are closely related to Child is CFLP^TMThe analysis does not show any divergence yet.   The above result is^TMUsing the assay, (16SrRN used as substrate) Distinguish between bacterial genera and different species and subspecies (depending on the region of the A gene) be able to. Same or similar genera (eg, Salmonella, Shigella) (Shigella) and CFLP produced in E. coli)^TMCompare patterns Then the band patterns are generally similar and the difference is a small subset It can be seen that it is shown as a change in band. Different genera (eg, E. coli (E . coli) and S. coli. aureus), the barcode pattern A more pronounced change is evident in CFLP^TMUsing pattern Not only can we detect differences between organisms, but also It is possible to evaluate the degree of branching in evolution between organisms.   CFLP^TMSubstrates for analysis can be used for PCR amplification using various primer sets Produced. Some primer pairs are used for all prokaryotes Reported as universal, other primer pairs were It has been found to be specific (see PCT Publication No. 90/15157). ply The DNA sequence of the rRNA gene from a particular organism, excluding the Knowledge of bacterial 16S rRNA gene amplification and CFLP^TMNot required for analysis.   Typical archaea and eubacteria, different phyla of eubacteria, different phyla within eubacteria , Different subphylums of the same phyla, different genera of the same group, different species of the same genus and different species of the same species Different CFLP among different strains^TMA pattern was observed. CFLP^TMPa Distinct features in the turn are related to food poisoning, the harm of normal flora Allowed to distinguish pathogenic isolates such as those from non-members Was done.   Using the same primer set and genomic DNA from various organisms PCR products were transferred during gel electrophoresis (polyacrylamide gel without gradient). Cleavase, which can not be distinguished by mobility^TMBN enzyme Cleaves these PCR products into shorter fragments, thereby producing a characteristic cleavage product Object (ie clear CFLP^TMCharacteristic). Cleavage products generated Patterns are reproducible and use given primer pairs in independent PCR DNA substrates made from the same organism will give the same pattern of cleavage products.   CFLP^TMThe pattern is for large DNA fragments (eg, at least about 1.6 kb). And thus cover the entire length of the bacterial 16S rRNA gene. -Can be. Also, CFLP^TMIs the entire 16S rRNA gene Can be used with shorter DNA fragments (about 200 bp) at different positions You.                                 Example 35                CFLP ^™ analysis of substrates containing nucleotide analogs   CFLP by using various nucleotide analogs^TMReaction substrate production The effect was investigated. As discussed below, nucleotide analogs can be used for several reasons. More used in PCR. Therefore, CFLP^TMModification of PCR by analysis That is, the analytical ability of the nucleotide analog-containing PCR product) was examined. 7-Deazap Phosphorus analogs (7-deaza-dATP and 7-deaza-dGTP) have multiple adjacent Destabilizes regions of secondary structure by weakening purine intrachain stacking Act like so. This effect allows the use of natural dNTPs for strong secondary structure Allows amplification of nucleic acids that are resistant to amplification [McConlogue et al., Nucleic  Acids Res., 16:20 (1988)].   Similarly, the analog dUTP is often used instead of dTTP, For another reason. dUTP-containing DNA (this nomenclature is derived from using dUTP). Shorthand name for the resulting PCR product. Actual PCR product contains dUMP . ) Can be destroyed by the enzymatic activity of uracil DNA glycosylase (UDG). However, the dTTP-containing DNA remains intact. PCR products are used in place of dTMP In addition, if UDG is used to produce dUMP, the use of UDG will And does not interfere with amplification from normal DNA False positive results due to passing can be eliminated. This method is useful for performing PCR. Widely used in clinical laboratories, thus this method is useful for CFL classification of pathogens. P^TMWill be used in most clinical laboratories that use PCR with. Sand Chi, CFLP^TMAppropriately cleave the dUTP-containing DNA fragment of the reaction (ie, , Producing a reproducible band pattern).   For these comparisons, the wild-type and R422Q mutations of the human tyrosinase gene Substrates corresponding to the 157 bp fragment derived from the exon of the variant were prepared using 1) dNTP (ie, A standard mixture of dATP, dCTP, dGTP and dTTP); 2) dT DUTP for TP; 3) 7-deaza-dGTP for dGTP (d⁷ GTP); and 4) 7-deaza-dATP (d instead of dATP)⁷ATP) Were made by PCR amplification using either These substrates are then Cleavase^TMIncubate with BN enzyme and The effect of the presence of the nucleotide analog on the cleavage pattern was investigated. B) Preparation of substrates containing nucleotide analogues   The 157 bp fragment (exon 4) of the human tyrosinase gene was replaced with 5'CACCGTCCTCTTCAAGA AG3 '(SEQ ID NO: 29) and 5' biotin-CTGAATCTTGTAGATAGCTA3 '(SEQ ID NO: Amplified by PCR using a pair of 30). Wild type of tyrosinase gene or A plasmid containing cDNA from the R422Q mutant was used as a template ( See Example 8 for a description of these plasmids). Double-stranded PCR obtained The product contains a 5 'biotin label on the antisense strand, so CFLP^TMDetect by reaction The sequence issued is SEQ ID NO: 35 (wild-type antisense strand) or SEQ ID NO: 53 ( R422Q mutant antisense strand). All PCRs are in a final volume of 100 μl went. dATP, dCTP, dGTP, dTTP and dUTP are available from Perkin E Obtained from lmer. d⁷ATP and d⁷GTP was obtained from Pharmacia. Taq  DNA polymerase was obtained from Promega. The PCR mixture is shown in Table 7 below. It was prepared as follows.   The wild-type and mutant R422Q substrates are replaced with the native and substituted nucleosides listed above. Amplification was performed using a tide analog. Natural dNTP, d⁷ATP and d⁷GTP In the case of the containing reaction solution, all the reaction components were added together. The reaction solution containing dUTP is First prepared without polymerase (see below).   The prepared reaction solution was used in a thermal cycler (MJ Research, Watertown, M.P.) preheated to 95 ° C. A). The tubes were incubated at 95 ° C. for 1 minute before amplification. The program was then cycled at 94 ° C for 30 minutes, 50 ° C for 1 minute and 72 ° C for 2 minutes for 34 cycles. Finally, the 72 ° C. incubation was set for 5 minutes.   Reactions containing dUTP were performed by "hot start". Polymerase All but the components were mixed, heated to 95 ° C for 1 minute, and then cooled to 72 ° C. Then, Taq Add polymerase (2.5 units) to 10 μl IX PCR buffer and add final volume To 100 μl.   At the end of the amplification, the PCR product, excluding the reaction solution containing dUTP, was O_FourAc solution and dUTP-containing reaction solution was 2M NH_FourAn OAc solution was used. Then All were precipitated by the addition of 2.5 volumes (total aqueous volume) of absolute ethanol. D The NA pellet was collected by centrifugation and dried under reduced pressure. 10 μl of pellet TE and 10 μl STOP buffer (20 μl T10E for dUTP containing reaction) 0.1 and 16 μl STOP). Then heat the test tube to 85 ° C for 2 minutes The mixture was then diluted to 10% using 7M urea in 0.5X TBE buffer (for dUTP). Is 6%) denaturing acrylamide gel (19: 1 cross-linking) Developed by electrophoresis.   PCR products corresponding to the 157 bp substrate from wild-type and R422Q mutants were Gel purification was performed as described in Example 19. The gel-purified DNA was Resuspended in 0E0.1 buffer. That is, 40 μl for a fragment containing only dNTPs; d⁷40 μl for fragments containing ATP; d⁷25 μl for fragments containing GTP; and And 25 μl for the fragment containing dUTP. B) Cleavage reaction conditions   Gel purified, containing natural deoxynucleotides and nucleotide analogs The 157 bp tyrosinase substrate was analyzed in a cleavage reaction as follows. Final reaction mix The material contained 1 μl of resuspended gel-purified DNA in a 20 μl volume (see section (a) above), 0.1 μl. 2 mM MnCl_Two25 ng of Cleavase (Clearase) in 10 mM MOPS, pH 7.5 containing vase)^TMContains BN, and 0.05% each of Tween 20 and NP-40. Enzyme-free pair Teru is Cleavase^TMIt was prepared by replacing the BN enzyme with distilled water. Substrate Distribute the DNA into reaction tubes and add H_TwoThe volume was adjusted to 15 μl with O. The rest of the reaction The components were mixed to a volume of 5 μl (ie, a 4 × concentration). DNA at 95 ° C for 15 seconds Heating denatured the DNA. The cleavage reaction was performed with 5 μl of the enzyme / buffer mixture (the 4 × Concentration). Incubate the cleavage reaction at 45 ° C for 3 minutes. The reaction was stopped by the addition of 16 μl of STOP solution (described in section (a)). 7 μl of each sample After heating the sample to 85 ° C. for 2 minutes, 45 mM trisborate, pH 8.3, 1.4 mM E 10% denaturing acrylamide gel using 7M urea in DTA buffer (19: 1 Bridge). For the gel, bromophenol blue transfers the length of the gel. Run at a constant 800V until run.   After electrophoresis, the biotinylated fragment was replaced with 4 μl of SAAP conjugate in 100 μl. Described in Example 8 except that it was added to USB shielding buffer (1: 25,000 dilution). Detected as listed. After washing, 5 μl of CDP-Star^TMAs a chemiluminescent substrate Used. FIG. 88 shows the obtained autoradiogram.   In FIG. 88, the lane marked "M" is Amersham (Arlington Heights, IL) 50, 100 and 200 nucleosides, including biotinylated molecular weight markers obtained from Includes bands corresponding to the length of the tide (size uses numbers and large arrowheads) Shown. ). Lanes 1-8 are Cleavase^TMIn the absence of BN enzyme Includes reaction products obtained by incubating the substrate with No or uncut control). Lanes 9-16 are cleaved (Cleavase)^TMReaction products obtained by incubating substrates in the presence of the BN enzyme Including things. Lanes 1, 3, 5, 7, 9, 11, 13 and 15 show wild-type substrate. Including. Lanes 2, 4, 6, 8, 10, 12, 14 and 16 have R422Q mutations Including body substrate. The products shown in lanes 1, 2, 9, and 10 were obtained by PCR with dNTPs. Produced from a substrate made using. Products shown in lanes 3, 4, 11, and 12 Was generated from a substrate made using dUTP instead of dTTP in PCR . The products shown in lanes 5, 6, 13 and 14 were replaced by dGTP instead of dGTP by PCR.⁷ It was generated from a substrate made using GTP. Lanes 7, 8, 15, and 16 The product shown in (d) was replaced by dATP in PCR.⁷Substrate prepared using ATP Generated from. As is clear from this example, the modified DNA fragment was subjected to CFLP reaction. Suitable for cleavage at The band pattern varies considerably with these substitutions, Wild-type and R422Q mutant DNA are easily distinguishable in all cases. Can be   The present invention is not limited to a particular theory, but uses nucleotide analogs. Changes in the band pattern observed when Can be In all cases, especially with reference to 7-deazapurine, nucleotide analogs The use of will substantially alter the nature and stability of the intrachain fold generated during the cleavage reaction. It is thought to change to. As a result, the position of the cleavage site naturally shifts. Sa In addition, replacement with modified nucleotides is required for the folded enzyme Change the affinity of the protein, enhancing or weakening cleavage at specific sites.   Found between wild-type and R422Q mutants when using various analogs Examination of the changes shows that the use of these substitutions can enhance the differences between the deformations. You. For example, two substrate DNAs (dUTP) in the region just above the 50 bp marker Or produced using dTTP) between wild type and mutant One significant band that decreases in intensity between, is more in the dU-containing sample. It greatly decreases.   The results shown in FIG. 88 show that CFLP was performed using nucleotide analogs.^TMMake the substrate Show that you can do it. Wildness of the tyrosinase gene containing nucleotide analogues Substrates derived from type- or R422Q mutants are mutant and wild-type alleles Generates distinct cleavage patterns that allow for gene differentiation and identification.   In this example, the exact site of cleavage is determined by the dNTP-containing substrate and 7-deaza-dN Depending on the TP containing substrate, dGTP may be 7-deaza-GTP or dATP. Clear CFLP pattern after replacing TP with 100% of 7-deaza-ATP Is generated. The above results also show that DNA fragments containing nucleotide analogs The single base change present in the nucleoside has a significant effect on the folded structure. DNA fragments containing no analog^TMWhen analyzed using the assay It shows that it causes a change in cleavage pattern similar to that seen.   As is apparent from the above, the present invention provides a rapid screening of nucleic acid sequences for mutations. Reagents and methods that allow for These methods are virus and Mutations that enable the identification of bacterial and bacterial pathogens and that are related to gene sequences (eg, For example, mutations or mutations associated with multidrug resistance in M. tuberculosis Mutations associated with the disease of These methods can help identify pathogens. Provide improved means for measurement and analysis.

────────────────────────────────────────────────── ─── Continuation of front page    (31) Priority claim number 08 / 484,956 (32) Priority date June 7, 1995 (33) Priority country United States (US) (31) Priority claim number 08 / 520,946 (32) Priority date August 30, 1995 (33) Priority country United States (US) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), AU, CA, JP, US (72) Liamichev, Victor Eye.             United States 53713 Wisconsin             State, Madison, Post Road # 5             2221 (72) Inventor Blow, Mary Andy.             United States 53711 Wisconsin             Hammersley Road, Madison, Oregon             5905 (72) Inventor Oldenburg, Mary C.             United States 53704 Wisconsin             State of Madison, North Third Street             Port 30 (72) Inventor Heisler, Laura M.             United States 53703 Wisconsin             , Madison, South Ingersoll               Street 610 (72) Inventor Fours, Lance             United States 91016 California             Cloverleaf Dora, Monrovia Province             Eve 1250 (72) Inventors Olive and David Michael             United States 53719 Wisconsin             State, Madison, Country Grove             Drive 3404

Claims (1)

[Claims] 1. A method for treating a nucleic acid, comprising: a) providing i) a cleavage means, and ii) a nucleic acid substrate; b) treating the substrate under conditions such that the nucleic acid substrate produces one or more cleavage structures. And c) reacting the cleavage means with the cleavage structure to obtain one or more cleavage products. 2. 2. The method according to claim 1, wherein said cleavage means is an enzyme. 3. 3. The method according to claim 2, wherein said enzyme is a nuclease. 4. Wherein nucleases cleave DNase (Cleavase) ^TM BN enzyme, Thermus aquaticus (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Thermus thermophilus) DNA polymerase, E. coli (Escherichia coli) Exo III, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) 4. The method of claim 3, wherein the method is selected from the group consisting of a Rad1 / Rad10 complex. 5. 2. The method of claim 1, wherein said nucleic acid substrate comprises a nucleotide analog. 6. 6. The method of claim 5, wherein said nucleotide analog is selected from the group consisting of 7-deaza-dATP, 7-deaza-dGTP and dUTP. 7. 2. The method of claim 1, wherein the nucleic acid of step (a) is essentially single-stranded. 8. 2. The method of claim 1, wherein said nucleic acid is RNA. 9. 2. The method according to claim 1, wherein said nucleic acid is DNA. Ten. The method according to claim 1, wherein the nucleic acid in step (a) is double-stranded. 11． The treatment of step (b) comprises: i) making the double-stranded nucleic acid essentially single-stranded, and ii) transforming the single-stranded nucleic acid under conditions such that the single-stranded nucleic acid has a secondary structure. 11. The method of claim 10, comprising exposing. 12． 12. The method of claim 11, wherein said double-stranded nucleic acid is made essentially single-stranded by increasing the temperature. 13. The method of claim 1, further comprising detecting the one or more cleavage products. 14. 2. The method of claim 1, wherein said nucleic acid substrate comprises an oligonucleotide comprising a human p53 gene sequence. 15． A method for treating nucleic acid, comprising: a) i) cleavage means in a solution containing manganese, and ii) providing a nucleic acid substrate; b) treating the nucleic acid substrate at an elevated temperature; Lowering the temperature under conditions such that a cleavage structure is formed, d) reacting the cleavage means with the cleavage structure to obtain one or more cleavage products, and e) one or more cleavage products. Detecting an object. 16． 16. The method according to claim 15, wherein said cleavage means is an enzyme. 17． 17. The method of claim 16, wherein said enzyme is a nuclease. 18． Wherein nucleases cleave DNase (Cleavase) ^TM BN enzyme, Thermus aquaticus (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Thermus thermophilus) DNA polymerase, E. coli (Escherichia coli) Exo III, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) 18. The method according to claim 17, wherein the method is selected from the group consisting of a Rad1 / Rad10 complex. 19. 16. The method of claim 15, wherein said nucleic acid substrate comprises a nucleotide analog. 20. 20. The method of claim 19, wherein said nucleotide analog is selected from the group consisting of 7-deaza-dATP, 7-deaza-dGTP and dUTP. twenty one. 16. The method according to claim 15, wherein said nucleic acid is RNA. twenty two. 16. The method according to claim 15, wherein said nucleic acid is DNA. twenty three. 16. The method of claim 15, wherein said nucleic acid of step (a) is double-stranded. twenty four. 16. The method according to claim 15, wherein said nucleic acid of step (a) is single-stranded. twenty five. 16. The method of claim 15, wherein said nucleic acid substrate comprises an oligonucleotide comprising a human p53 gene sequence. 26. 16. The method of claim 15, wherein said nucleic acid substrate comprises an oligonucleotide comprising a microbial gene sequence. 27. A method for detecting a mutation in the human p53 gene, comprising: a) i) a cleavage means, and ii) a nucleic acid substrate containing a human p53 gene sequence, and b) a nucleic acid substrate comprising one or more cleavage structures. Treating said substrate under conditions such as to produce c) reacting said cleavage means with said cleavage structure to obtain one or more cleavage products, and d) cleaving said cleavage products with a reference p53 gene sequence Comparing with the cleavage product obtained by the method. 28. The cleavage product obtained by cleavage of the reference p53 gene sequence is generated by cleavage of a nucleic acid substrate containing a human p53 gene sequence selected from the group consisting of SEQ ID NOs: 79-81, 84-89 and 94-97. The method according to 27. 29. A method for identifying a strain of a microorganism, comprising the steps of: a) providing i) cleavage means, and ii) a nucleic acid substrate containing a sequence derived from one or more microorganisms; Treating the substrate under conditions that produce it, and c) reacting the cleavage means with the cleavage structure to obtain one or more cleavage products. 30. 30. The method of claim 29, wherein said cleavage means is an enzyme. 31. 31. The method of claim 30, wherein said enzyme is a nuclease. 32. Wherein nucleases cleave DNase (Cleavase) ^TM BN enzyme, Thermus aquaticus (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Thermus thermophilus) DNA polymerase, E. coli (Escherichia coli) Exo III, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) 32. The method according to claim 31, wherein the method is selected from the group consisting of a Rad1 / Rad10 complex. 33. 30. The method of claim 29, wherein said nucleic acid substrate comprises a nucleotide analog. 34. 34. The method of claim 33, wherein said nucleotide analog is selected from the group consisting of 7-deaza-dATP, 7-deaza-dGTP and dUTP. 35. 30. The method of claim 29, wherein said nucleic acid of step (a) is essentially single-stranded. 36. 30. The method of claim 29, wherein said nucleic acid is RNA. 37. 30. The method according to claim 29, wherein said nucleic acid is DNA. 38. 30. The method of claim 29, wherein said nucleic acid of step (a) is double-stranded. 39. The treatment of step (b) comprises: i) making the double-stranded nucleic acid essentially single-stranded, and ii) transforming the single-stranded nucleic acid under conditions such that the single-stranded nucleic acid has a secondary structure. 39. The method of claim 38, comprising: exposing. 40. 40. The method of claim 39, wherein increasing the temperature renders the double-stranded nucleic acid essentially single-stranded. 41. 30. The method of claim 29, further comprising detecting the one or more cleavage products. 42. 30. The method of claim 29, wherein said microorganism comprises a bacterium. 43. The bacterium is a member of the genus Campylobacter (Campylobacter), Eschericia (Escheric hia), Mycobacterium (Mycobacterium), Salmonella (Salmonella), Shigella (Shigella) and Staphylococcus (Staphylococcus). 43. The method of claim 42, which is selected. 44. 44. The method of claim 43, wherein the member of the genus Mycobacterium comprises a strain of multidrug-resistant Mycobacterium tuberculosis. 45. 30. The method of claim 29, wherein said microorganism comprises a virus. 46. 46. The method of claim 45, wherein said virus is selected from the group consisting of hepatitis C virus and simian immunodeficiency virus. 47. A method for detecting and identifying a strain of a microorganism, comprising: a) extracting nucleic acid from a sample suspected of containing one or more microorganisms; and b) combining the extracted nucleic acid and cleavage means with one or more of the extracted nucleic acids. And contacting under conditions such that the cleavage means cleaves the secondary structure to yield one or more cleavage products. 48. 48. The method of claim 47, further comprising separating the cleavage product. 49. 50. The method of claim 47, further comprising detecting the cleavage product. 50. Further comprising comparing a detected cleavage product obtained from cleavage of the extracted nucleic acid isolated from the sample with a cleavage product isolated from cleavage of nucleic acid derived from one or more reference microorganisms. 50. The method of claim 49. 51. After the extraction in step a), isolating the polymorphic locus from the extracted nucleic acid to obtain a nucleic acid substrate comprising a sequence derived from the polymorphic locus, and contacting the substrate with the cleavage means in step b); 50. The method of claim 47. 52. 52. The method of claim 51, wherein said polymorphic locus is isolated by nucleic acid amplification. 53. 53. The method of claim 52, wherein said amplification is performed in the presence of a nucleotide analog. 54. 54. The method of claim 53, wherein said nucleotide analog is selected from the group consisting of 7-deaza-dATP, 7-deaza-dGTP, and dUTP. 55. 53. The method of claim 52, wherein said amplification uses oligonucleotide primers that match a consensus gene sequence from said polymorphic locus. 56. 53. The method of claim 52, wherein said amplification uses oligonucleotide primers complementary to a consensus gene sequence from said polymorphic locus. 57. 52. The method of claim 51, wherein said polymorphic locus comprises a ribosomal RNA gene. 58. 58. The method of claim 57, wherein said ribosomal RNA gene is a 16S ribosomal RNA gene. 59. 48. The method of claim 47, wherein said cleavage means is an enzyme. 60. 60. The method of claim 59, wherein said enzyme is a nuclease. 61. Wherein nucleases cleave DNase (Cleavase) ^TM BN enzyme, Thermus aquaticus (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Thermus thermophilus) DNA polymerase, E. coli (Escherichia coli) Exo III, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) 61. The method of claim 60, wherein the method is selected from the group consisting of a Rad1 / Rad10 complex. 62. 50. The method of claim 47, wherein said nucleic acid of step (a) is essentially single-stranded. 63. 48. The method of claim 47, wherein said nucleic acid is RNA. 64. 48. The method of claim 47, wherein said nucleic acid is DNA. 65. 48. The method of claim 47, wherein said nucleic acid of step (a) is double-stranded. 66. The treatment of step (b) comprises: i) making the double-stranded nucleic acid essentially single-stranded, and ii) transforming the single-stranded nucleic acid under conditions such that the single-stranded nucleic acid has a secondary structure. 66. The method of claim 65, comprising exposing. 67. 67. The method of claim 66, wherein increasing the temperature renders the double-stranded nucleic acid essentially single-stranded. 68. 50. The method of claim 47, wherein said microorganism comprises a bacterium. 69. The bacterium is a member of the genus Campylobacter (Campylobacter), Eschericia (Escheric hia), Mycobacterium (Mycobacterium), Salmonella (Salmonella), Shigella (Shigella) and Staphylococcus (Staphylococcus). 69. The method of claim 68, which is selected. 70. 70. The method of claim 69, wherein the member of the genus Mycobacterium comprises a multidrug-resistant strain of Mycobacterium tuberculosis. 71. 50. The method of claim 47, wherein said microorganism comprises a virus. 72. 72. The method of claim 71, wherein said virus is selected from the group consisting of hepatitis C virus and simian immunodeficiency virus. 73. A nucleic acid processing kit, comprising: a) an enzyme that reacts with a cleavage structure to generate a cleavage product; and b) a solution containing manganese. 74. 74. The kit of claim 73, further comprising a reagent for detecting said cleavage product. 75. 75. The kit of claim 74, wherein said enzyme is a nuclease. 76. Wherein nucleases cleave DNase (Cleavase) ^TM BN enzyme, Thermus aquaticus (Thermus aquaticus) DNA polymerase, Thermus thermophilus (Thermus thermophilus) DNA polymerase, E. coli (Escherichia coli) Exo III, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) 76. The kit of claim 75, wherein the kit is selected from the group consisting of a Rad1 / Rad10 complex.