JP5661071B2 - RNA detection method - Google Patents

Description

  This application is a continuation-in-part of U.S. Patent Application Nos. 09 / 577,304 and 09 / 758,282, and is a continuation-in-part of U.S. Patent Application No. 09 / 350,309, a divisional application of U.S. Patent No. 5,985,557. Yes, a PCT application US 98/05809 domestic application, a continuation-in-part of co-pending U.S. Patent Application No. 09 / 381,212, U.S. Patent Nos. 5,994,069, 6,090,543, It claims priority to 5,985,557, 6,001,567 and 5,846,717, and PCT application US 97/01072.

The present invention relates to nucleic acid sequences and compositions and methods for detecting and characterizing mutations in nucleic acid sequences. The present invention relates to a method for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, the 5 ′ nuclease activity of various enzymes is used to cleave target-dependent cleavage structures, thereby causing specific nucleic acid sequences or specific nucleic acid sequence variations. Indicate that.

BACKGROUND OF THE INVENTION Methods for detecting and characterizing specific nucleic acid sequences and sequence variations include the detection of the presence of nucleic acid sequences that are indicative of viral or bacterial infections, and gene mutations or alleles associated with disease or cancer. It is used to detect the presence of a gene. These methods can also be applied when identifying the owner of a nucleic acid for forensic analysis or parentage testing.

Various methods are known in the art that can be used to detect and characterize specific nucleic acid sequences and sequence variations. Nonetheless, as nucleic acid sequencing of the human genome, as well as the genomes of many pathogenic organisms, has been completed, there is a need for specific nucleic acid sequence detection assays that are fast, reliable, inexpensive, and convenient. Continues to grow. Importantly, these test methods must be able to generate a detectable signal from a sample that contains very little of the sequence of interest. The following discussion considers the following nucleic acid detection assays currently in use on two criteria: Signal amplification technology for detection of trace sequences; II. Direct detection techniques for quantitative detection of sequences; and III. Direct detection of RNA.
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I. Signal amplification techniques for amplification "Polymerase chain reaction" (PCR) is the first generation of nucleic acid amplification methods. However, although the basis for specificity is the same, several other methods have been developed to generate signals by different amplification mechanisms. Such methods include “ligase chain reaction” (LCR), “Self-Sustained Synthetic Reaction” (3SR / NASBA), and “Qβ-replicase” (Qβ).

Polymerase chain reaction (PCR)
U.S. Pat. Nos. 4,683,195 (Patent Document 1), 4,683,202 (Patent Document 2), and 4,965,188 (Patent Document 3) granted to Mullis and Mullis et al. (The disclosures of which are incorporated herein by reference) The polymerase chain reaction (PCR) describes a method for increasing the concentration of a segment of a target sequence in a genomic DNA mixture without cloning or purification. This technique provides a way to solve the problem of low target sequence concentrations. Using PCR, the concentration of the target can be directly raised to an easily detectable height. This target sequence amplification method involves introducing a molar mixture of two types of oligonucleotide primers complementary to each strand of a double-stranded target sequence into a DNA mixture containing the desired target sequence. This mixture is denatured and then hybridized. After hybridization, the primer is extended by polymerase to form a complementary strand. To obtain the desired target sequence segment at a relatively high concentration, the steps of denaturation, hybridization, and polymerase extension are repeated as necessary.

  Since the length of the desired target sequence segment can be measured from the relative position of the primers to each other, this length is an adjustable parameter. These are said to be “PCR amplified” because the desired target sequence segments become dominant sequences (in terms of concentration) in the mixture.

Ligase chain reaction (LCR or LAR)
Ligase chain reaction (LCR; Barany, Proc. Natl. Acad. Sci., 88: 189 (1991) (Non-patent document 1); Barany, PCR Methods and Applic., 1: 5 (1991) (Non-patent document 2) As well as Wu and Wallace, Genomics 4: 560 (1989) (also referred to as ligase amplification reaction (LAR)) is a well-recognized alternative nucleic acid amplification method. It has become. In LCR, four types of oligonucleotides are mixed: two adjacent oligonucleotides that hybridize specifically to one strand of the target DNA, and a complementary set of adjacent oligonucleotides that hybridize to the opposite strand. Add DNA ligase to the mixture. If there is complete complementarity at the binding site, the ligase covalently binds each set of hybridized molecules. The key to LCR is that the two probes are linked together only when they base pair with the sequence in the target sample without gaps or mismatches. By repeating the cycle of denaturation, hybridization, and ligation, short segments of DNA are amplified. In addition, enhanced detection of single nucleotide mutations has been achieved using LCR in combination with PCR. Segev, PCT International Publication No. 9001069A1 (1990) (Patent Document 4). However, the four oligonucleotides used in this assay can pair together to form two short ligable fragments, which can generate a background signal independent of the target . The use of LCR for mutation screening is limited only when examining specific nucleic acid positions.

Independent synthesis reaction (3SR / NASBA)
Self-supporting synthesis reaction (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87: 1874-1878 [1990] (Non-Patent Document 4), Correction of errors, Proc. Natl. Acad. Sci., 87: 7797 [1990] (Non-Patent Document 5) is a transcription-based in vitro amplification system that can amplify RNA sequences logarithmically at a uniform temperature (Kwok et al., Proc. Natl. Acad. Sci. 86: 1173-1177 [1989] (non-patent document 6)). The amplified RNA can be used for mutation detection (Fahy et al., PCR Meth. Appl., 1:25 [1991] (Non-patent Document 7)). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5 ′ end of the target sequence. In a mixture of enzyme and substrate, including another primer, reverse transcriptase, RNase H, RNA polymerase, and ribo- and deoxyribo-nucleoside triphosphates, transcription of the target sequence to amplify the region of interest , CDNA synthesis, and second strand synthesis are repeated. The use of 3SR for mutation detection is kinetically limited to screening small segments of DNA (eg, 200-300 base pairs).

Q-beta (Qβ) replicase In this method, a probe that recognizes the sequence of interest binds to a template RNA that is replicable for Qβ replicase. The major problem of false positives that resulted from replicating non-hybridized probes was previously identified, but this problem is addressed by employing a sequence-specific ligation process. However, available thermostable DNA ligases are not effective for this RNA substrate and must be ligated at low temperatures (37 ° C.) with T4 DNA ligase. For this reason, high temperature cannot be used as a means for realizing the same specificity as LCR, and a ligation reaction can be used to detect a mutation at the binding site, but not for other sites.

  Table 2 below lists some desirable features for systems useful in sensitive nucleic acid diagnostics and summarizes the capabilities of each of the major amplification methods (or Landgren, Trends in Genetics 9: 199). [1993] (see Non-Patent Document 8)).

  A suitable diagnostic method needs to be very specific. A simple way to control nucleic acid hybridization is to adjust the reaction temperature. The 3SR / NASBA and Qβ systems can all produce large amounts of signal, but one or more of the enzymes contained in each cannot be used at high temperatures (ie,> 55 ° C.). Shortening the probe so that it can be easily melted even at low temperatures increases the likelihood that anything other than a perfect sequence match in a complex genome will occur. For these reasons, PCR and LCR have recently become major in the field of detection technology research.

(Table 1)

The basis for amplification methods in PCR and LCR is the fact that the product in one cycle can be used as a template in all subsequent cycles, resulting in a doubling of the population with each cycle. The final yield in such a doubling system can be expressed as (1 + X) ⁿ = y. Where “X” is the average efficiency (% replicated in each cycle), “n” is the number of cycles and “y” is the overall efficiency, ie the yield of the reactant (Mullis , PCR Methods Applic., 1: 1 [1991] (Non-Patent Document 9)). If all copies of the target DNA were used as templates in all cycles of the polymerase chain reaction, the average efficiency is 100%. If performed 20 cycles of PCR, yield is 2 ²⁰ times the starting material, that is, 1,048,576 copies. Decreasing the reaction conditions to an average efficiency of 85% results in 1.85 ²⁰ times the starting material, ie 220,513 copies, in 20 cycles as well. In other words, when PCR is performed at an efficiency of 85%, only 21% of the final product can be obtained compared to when the reaction is performed at an efficiency of 100%. When the reaction is reduced to an average efficiency of 50%, yields of less than 1% of possible products are obtained.

  In fact, the normal polymerase chain reaction rarely achieves the theoretical maximum yield, so PCR is usually performed for 20 cycles or more to compensate for the low yield. At an average efficiency of 50%, it is theoretically impossible to achieve a million-fold amplification that is possible in 20 cycles unless 34 cycles are performed. And at lower efficiencies, the number of cycles required is inconsequential. It should be noted that the background product becomes the major product when amplified with higher average efficiency than the target of interest.

  Also, to name just a few, various variables affect the average efficiency of PCR, including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition. Contamination of the reaction solution by exogenous DNA (for example, DNA spilled into laboratory equipment), that is, cross contamination is also an important examination condition. Reaction conditions need to be carefully optimized for each primer pair and target sequence, and even a skilled researcher can take this process for days. The complexity of this process, including numerous technical considerations and other factors, greatly hinders the use of PCR in clinical settings. In fact, PCR has not yet penetrated the medical market dramatically. The same problem arises with LCR because the LCR must be optimized to use different oligonucleotide sequences for each target sequence. Furthermore, both methods require expensive equipment that can carry out accurate temperature cycles.

  Many nucleic acid detection techniques, such as when investigating allelic variations, not only detect specific sequences in a complex background, but also differ by only a few or a single nucleotide difference. Including the distinction. One method of detecting allele-specific mutations by PCR is based on the fact that it is difficult for Taq polymerase to synthesize a DNA strand if there is a mismatch between the template strand and the 3 'end of the primer. Yes. By using primers that perfectly match only one of the possible alleles, allele-specific mutations can be detected. The sequence is not amplified because a mismatch with another allele prevents extension of the primer. This method has considerable limitations in that the base composition of the mismatch sequence affects the possibility of inhibiting the extension of the mismatch sequence, and that certain mismatches do not prevent or have minimal effect on the extension ( Kwok et al., Nucl. Acids Res., 18: 999 [1990] (Non-Patent Document 10)).

  A similar 3'-mismatch method has been used to inhibit ligation in LCR with higher effectiveness (Barany, PCR Methods and Applic., 1: 5 (1991)). Any mismatch effectively blocks the action of the thermostable ligase, but the problem with LCR is that the product of target-independent background ligation initiates amplification. Furthermore, combining PCR followed by LCR to identify nucleotides at individual positions is clearly a cumbersome plan for clinical studies.

II. Direct detection techniques If a sufficient amount of nucleic acid to be detected can be used, it is advantageous to be able to detect the sequence directly without making more copies of the target (eg, with PCR and LCR). is there. Most notably, the method that does not multiply the signal logarithmically is more sensitive to quantitative analysis. Even if multiple dyes are attached to one nucleotide to enhance the signal, the correlation between the final signal intensity and the target amount is straightforward. Such a system has the further advantage that contamination of the surface of the laboratory instrument by the product is not of great interest since the reaction product itself does not proceed further. Traditional direct detection methods, such as Northern and Southern blotting, and RNase protection assays, are not amenable to automation because they usually require the use of radioactivity. Recently devised techniques have aimed to eliminate the use of radioactivity and / or increase sensitivity in an automated manner. Two examples are the “Cycling Probe Reaction” (CPR) and the “Branched DNA” (bDNA).

  In the cycling probe reaction (CPR) (Duck et al., BioTech., 9: 142 [1990]), the long chimera is composed of RNA at the center and DNA at the two ends. Use oligonucleotides. Hybridization of the probe to the target DNA and exposure to the thermostable RNase H results in degradation of the RNA portion. This destabilizes the remaining double-stranded DNA portion and dissociates the remaining probe from the target DNA, allowing another probe molecule to repeat this process. A signal in the form of a cleaved probe molecule accumulates at a linear rate. If this process is repeated, the signal increases, but the RNA site of the oligonucleotide is easily degraded by the RNase introduced by sample preparation.

  The branched DNA method (bDNA) described by Urdea et al., Gene 61: 253-264 (1987) (13) provides each oligonucleotide with 35 to 40 labels (eg, alkaline phosphatase enzyme). Including an oligonucleotide having a branched structure. This enhances the signal produced by hybridization, but also enhances the signal produced by non-specific binding.

  Both of these methods have the advantages of the direct detection method described above, but both CPR and bDNA methods have two or more types of probes that are common in the signal amplification method described in Section I above ( It is not possible to take advantage of the specificity that is possible by requiring separate recognition by oligonucleotide) sequence. The requirement that two oligonucleotides must bind to one target nucleic acid in order to generate a detectable signal provides a special stringency criterion for any detection assay. The need for two oligonucleotides to bind to one target nucleic acid reduces the chance that a false “positive” result will result from non-specific binding of the probe to the target. In addition, as required in PCR, the oligonucleotide must be oriented in the opposite but correct direction so that the DNA polymerase can fill the gap between the two oligonucleotides from both directions, The requirement that the two oligonucleotides must be bound in a specific direction to the target further enhances the specificity of the detection reaction. However, while PCR uses two oligonucleotide probes (called primers), “non-specific” amplification (ie, sequence amplification not directed by the two primers used) is a common artifact. This is well known to those skilled in the art. This is due in part to the fact that DNA polymerases used for PCR can cope with even very long distances between oligonucleotides, as measured by nucleotides. This is because there is a large frame that can lead to a logarithmic increase. In contrast, LCR cannot allow the reaction to proceed unless the oligonucleotides used are bound next to each other. For this reason, the benefits of hybridization with double oligonucleotides are well recognized.

  An ideal direct detection method would combine the advantages of a direct detection assay (eg ease of quantification, minimal risk of carry-over contamination) with a double oligonucleotide hybridization assay. It is done.

III. RNA Direct Detection Methods In molecular medicine, simple and inexpensive methods for detecting RNA directly and quantitatively will greatly facilitate RNA virus analysis and measurement of specific gene expression. Both of these issues are now urgent issues in the field. Despite this need, few truly direct technologies have emerged. Detection assays that utilize PCR require RNA to be converted to DNA before amplification or introduction of variables that can compromise accurate quantification. In addition, PCR and other methods based on logarithmic amplification (eg NASBA) require hand-held containment devices to prevent cross-contamination, and small quantitative differences (eg 2 to 3 times). It is difficult to distinguish. Other test methods that examine RNA directly have a variety of disadvantages, including time-consuming autoradiographic steps (eg, RNase protection assays) and overnight reaction times (eg, branched DNA assays). There is. In the United States, more than 1.5 million virus loads are measured each year, so there is great potential for an inexpensive, fast, and high-throughput system for quantitative RNA measurements.

  Techniques for directly and quantitatively detecting mRNA are important for observing the expression of many different genes. In particular, expression levels of cytokines (eg, interleukins and lymphokines) have been used as a clinical measure of immune response in a wide range of disease progression (Van Deuren et al., J. Int. Fed. Clin. Chem., 5: 216 [ 1993] (Non-Patent Document 14), Van Deuren et al., J. Inf. Dis., 169: 157 [1994] (Non-Patent Document 15), Perenboom et al., Eur. J. Clin. Invest., 26: 159 [1996] (Non-patent document 16), Guidotti et al., Immunity 4: 25 [1996] (non-patent document 17)), and for monitoring transplant recipients (Grant et al., Transplantation 62: 910 [1996] (non-patent document). Reference 18)). In addition, monitoring viral load and identifying viral genotypes have clinical significance for people with viral infections caused by pathogens such as HIV and hepatitis C virus (HCV). There is a high correlation between viral load (ie, the absolute number of virus particles in the bloodstream) and the time to progress to AIDS (Mellors et al., Science 272: 1167 [1996] (Non-Patent Document 19). ), Saag et al., Nature Medicine 2: 265 [1996] (Non-Patent Document 20)). For this reason, viral load as measured by nucleic acid quantitative tests is becoming the standard monitoring tool for assessing the effectiveness and clinical status of HIV positive patients. It is considered important to reduce the viral load as early as possible and to regularly assess viral load. In the case of HCV, viral genotype has clinical significance because it correlates with the severity of liver disease and responsiveness to interferon therapy. Furthermore, because HCV cannot be cultured, new antiviral treatments can only be evaluated by establishing a correlation between characteristics such as viral genotype and clinical outcome.

  Although the above methods were useful for research or limited clinical applications when the throughput was low, large-scale quantitative analysis of RNA that can be easily applied to any gene system Clearly, the legal system requires more innovative efforts. An ideal direct detection method combines the advantages of a direct detection assay (eg, ease of quantification, minimal risk of carry-over contamination) with the specificity provided by dual oligonucleotide hybridization assays. It is considered to be a combination.

  Many of the methods described above rely only on hybridization to distinguish target molecules from other nucleic acids. Although some of these methods are very sensitive, it is almost impossible to accurately quantify or distinguish closely related mRNAs, particularly RNA that is expressed in different amounts in the same sample. While the above methods serve several purposes, there is a particular need for techniques that skillfully distinguish specific RNAs from closely related molecules.

U.S. Pat.No. 4,683,195 U.S. Pat.No. 4,683,202 U.S. Pat.No. 4,965,188 PCT International Publication No. 9001069A1

Barany, Proc. Natl. Acad. Sci., 88: 189 (1991) Barany, PCR Methods and Applic., 1: 5 (1991) Wu and Wallace, Genomics 4: 560 (1989) Guatelli et al., Proc. Natl. Acad. Sci., 87: 1874-1878 [1990] Guatelli et al., Proc. Natl. Acad. Sci., 87: 7797 [1990] Kwok et al., Proc. Natl. Acad. Sci., 86: 1173-1177 [1989] Fahy et al., PCR Meth. Appl., 1: 25 [1991] Landgren, Trends in Genetics 9: 199 [1993] Mullis, PCR Methods Applic., 1: 1 [1991] Kwok et al., Nucl. Acids Res., 18: 999 [1990] Barany, PCR Methods and Applic., 1: 5 (1991) Duck et al., BioTech., 9: 142 [1990] Urdea et al., Gene 61: 253-264 (1987) Van Deuren et al., J. Int. Fed. Clin. Chem., 5: 216 [1993] Van Deuren et al., J. Inf. Dis., 169: 157 [1994] Perenboom et al., Eur. J. Clin. Invest., 26: 159 [1996] Guidotti et al., Immunity 4: 25 [1996] Grant et al., Transplantation 62: 910 [1996] Mellors et al., Science 272: 1167 [1996] Saag et al., Nature Medicine 2: 265 [1996]

  The present invention relates to nucleic acid sequences and compositions and methods for detecting and characterizing mutations in nucleic acid sequences. The present invention relates to a method for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, the 5 ′ nuclease activity of various enzymes is used to cleave target-dependent cleavage structures, thereby causing specific nucleic acid sequences or specific nucleic acid sequence variations. Is shown.

  The present invention provides structure-specific cleavage factors (eg, nucleases) derived from a variety of biological sources, including mesophilic, chilling, thermophilic, and hyperthermophilic organisms. A preferred structure-specific nuclease is thermostable. Thermostable structure-specific nucleases are particularly useful in that nucleic acid hybridization acts at very specific temperatures, allowing for allele-specific detection (such as single base mismatches). In one embodiment, the thermostable structure-specific nuclease is a Thermus species such as, but not limited to, Thermus aquaticus, Thermus flavus, Thermus thermophilus, and the like. A thermostable 5 'nuclease containing a modified polymerase derived from a natural polymerase. However, the present invention is not limited to the use of thermostable 5 ′ nucleases. Also preferred are thermostable structure-specific nucleases derived from nucleases classified as FEN-1, RAD2, and XPG.

  The present invention is a method for detecting a target sequence (eg, mutation, polymorphism, etc.) that forms a invasive cleavage structure in the presence of a sample suspected of containing the target sequence, the target sequence. An oligonucleotide is provided, and a method is provided that includes providing an agent for detecting the presence of an invasive cleavage structure, and exposing the sample to the oligonucleotide and the agent. In some embodiments, the method further comprises detecting (directly or indirectly) a complex comprising an agent and an invasive cleavage structure. In some embodiments, the factor comprises a cleavage factor. In some preferred embodiments, the step of exposing the sample to the oligonucleotides and factors comprises subjecting the sample to conditions under which an invasive cleavage structure is formed between the target sequence and the oligonucleotide when the target sequence is present in the sample. Is exposed to oligonucleotides and factors to cleave this invasive cleavage structure with a cleavage factor to form a cleavage product. In some embodiments, the method further comprises detecting a cleavage product. In some embodiments, the target sequence comprises a first region and a second region, wherein the second region is present continuously downstream of the first region, and the oligonucleotide comprises the first and second regions Comprising an oligonucleotide, wherein at least a portion of the first oligonucleotide is completely complementary to the first site of the target sequence, and the second oligonucleotide comprises a 3 ′ site and a 5 ′ site, This 5 ′ site is completely complementary to the second site of the target nucleic acid.

  The present invention also provides a kit for detecting such a target sequence, comprising an oligonucleotide capable of forming an invasive cleavage structure in the presence of the target sequence. In some embodiments, the kit further comprises an agent (eg, a cleavage agent) for detecting the presence of an invasive cleavage structure. In some embodiments, the oligonucleotide comprises first and second oligonucleotides, the first oligonucleotide comprising a 5 ′ site complementary to the first region of the target nucleic acid, and the second The oligonucleotide comprises a 3 ′ site and a 5 ′ site complementary to the second region of the target nucleic acid present in succession downstream of the first region. In some preferred embodiments, the target sequence comprises.

  The present invention also provides a method for detecting the presence of a target nucleic acid molecule by detecting a non-target cleavage product, wherein the cleavage factor; the source of the target nucleic acid; the second region continues downstream from the first region. A target nucleic acid comprising a first region and a second region, wherein: at least a portion of the first oligonucleotide is completely complementary to the first site of the target sequence; and Providing a second oligonucleotide comprising a 3 ′ site and a 5 ′ site, wherein the 5 ′ site is completely complementary to the second site of the target nucleic acid; a cleavage factor, the target nucleic acid, the first oligonucleotide; And the second oligonucleotide are mixed so that at least a portion of the first oligonucleotide is annealed to the first region of the target nucleic acid and at least the 5 ′ site of the second oligonucleotide is the first of the target nucleic acid. Second territory A reaction mixture is formed under reaction conditions such that cleavage of the cleavage structure occurs to produce a non-target cleavage product; and detection of cleavage of the cleavage structure A method comprising:

  Detection of the cutting of the cutting structure can be performed by an arbitrary method. In some embodiments, detection of cleavage of the cleavage structure comprises detection of non-target cleavage products. In yet another aspect, detecting cleavage of the cleavage structure comprises detecting fluorescence, mass, or fluorescence energy transfer. Other detection methods include, but are not limited to, detecting radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge. In some embodiments, providing a protein comprising a non-targeted cleavage product, two types of single stranded nucleic acids that are annealed to define a single stranded portion of a protein binding region, and wherein the protein is a protein Detection is performed by a method comprising exposing a non-target cleavage product to a single-stranded portion of the protein binding region under conditions that bind to the binding region. In some embodiments, the protein comprises a nucleic acid generating protein that binds to a protein binding region to produce a nucleic acid. In some embodiments, the protein binding region is a template dependent RNA polymerase binding region (eg, a T7 RNA polymerase binding region). In another aspect, providing a non-target cleavage product, a single continuous nucleic acid strand comprising a sequence that defines a single-stranded portion of an RNA polymerase binding region, a template-dependent DNA polymerase, and a template-dependent RNA polymerase Exposing the non-target cleavage product to the RNA polymerase binding region under conditions that allow the non-target cleavage product to bind to a single-stranded portion of the RNA polymerase binding region to produce a bound non-target cleavage product, double-stranded RNA Exposing the bound non-targeted cleavage product to a template-dependent DNA polymerase under conditions that produce a polymerase binding region, and a template-dependent double-stranded RNA polymerase binding region under conditions such that an RNA transcript is generated. Detection is performed by a method comprising a step of exposing to RNA polymerase. In some embodiments, the method further comprises detecting an RNA transcript. In some embodiments, the template dependent RNA polymerase is T7 RNA polymerase.

  The present invention is not limited by the nature of the 3 ′ site of the second oligonucleotide. In some preferred embodiments, the 3 ′ site of the second oligonucleotide has a 3 ′ terminal nucleotide that is not complementary to the target nucleic acid. In some embodiments, the 3 ′ site of the second oligonucleotide comprises a single nucleotide that is not complementary to the target nucleic acid.

  Any component of the method can be attached to a solid support. For example, in some embodiments, the first oligonucleotide is attached to a solid support. In another embodiment, the second oligonucleotide is attached to a solid support.

  A cleavage factor is a factor that can cleave an invasive cleavage structure. In some embodiments, the cleavage factor comprises a structure specific nuclease. In particularly preferred embodiments, the structure-specific nuclease comprises a thermostable structure-specific nuclease (eg, a thermostable 5 ′ nuclease). A thermostable structure-specific nuclease contains an amino acid sequence that is homologous to part of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism (eg, Thermus aquaticus, Thermus flavus, and Thermus thermophilus). There are nucleotides that have, but are not limited to. In another embodiment, the thermostable structure-specific nuclease comprises a nuclease derived from a nuclease classified as FEN-1, RAD2, or XPG, or a portion of more than one of the above cleavage factors Includes chemical structure.

  The method is not limited by the nature of the target nucleic acid. In some embodiments, the target nucleic acid is single-stranded or double-stranded DNA or RNA. In some embodiments, the double stranded nucleic acid is made single stranded (eg, by heat) prior to forming the cleavage structure. In some embodiments, the source of the target nucleic acid includes a sample containing genomic DNA. Samples include, but are not limited to, blood, saliva, cerebrospinal fluid, pleural effusion, milk, lymph, sputum, and semen.

In some embodiments, the reaction conditions in the method include providing a source of divalent cations. In some embodiments, the divalent cation is selected from the group consisting of Mn ²⁺ ions and Mg ²⁺ ions. In some embodiments, the reaction conditions in the method include providing an excess concentration of the first and second oligonucleotides relative to the target nucleic acid.

  In some embodiments, the method further comprises a third oligonucleotide complementary to the third site of the target nucleic acid upstream of the first site of the target nucleic acid, and mixed with the reaction mixture. Providing a third oligonucleotide.

  The present invention also provides a method for detecting the presence of a target nucleic acid moiety by detecting a non-target cleavage product, wherein the cleavage factor; the source of the target nucleic acid; the second region is continuous downstream of the first region. A target nucleic acid comprising a first region and a second region, wherein a plurality of first oligonucleotides wherein at least a portion of the first oligonucleotide is completely complementary to the first site of the target sequence; And providing a second oligonucleotide comprising a 3 ′ site and a 5 ′ site, wherein the 5 ′ site is completely complementary to the second site of the target nucleic acid; a cleavage factor, the target nucleic acid, a plurality of first Mixing the oligonucleotide and the second oligonucleotide so that at least a portion of the first oligonucleotide is annealed to the first region of the target nucleic acid and at least the 5 ′ site of the second oligonucleotide is the target nucleic acid First The reaction conditions are such that a cleavage structure is formed by annealing to a region of the region, and further, cleavage of the cleavage structure occurs to generate a non-target cleavage product, and a plurality of types of cleavage structures can be formed. Providing a reaction mixture under reaction conditions such that it can be cut from; and detecting the cleavage of the cleavage structure. In some embodiments, the conditions include isothermal conditions that dissociate the plurality of first oligonucleotides from the target nucleic acid. While the present invention is limited by the number of cleavage structures formed on a particular target nucleic acid, in some embodiments, two or more of a plurality of first oligonucleotides (3, 4, 5, ..., 10, ..., 10000, ...) form cleavage structures with specific target nucleic acids and cleave these cleavage structures to produce non-target cleavage products.

The present invention also provides a method of using the cleavage product produced by the above method in yet another invasive cleavage reaction. For example, the present invention provides a cleavage factor; a first target nucleic acid comprising a first region and a second region, wherein the second region is continuously present downstream of the first region; A first oligonucleotide that is at least partially complementary to the first site of the first target nucleic acid; comprising a 3 ′ site and a 5 ′ site, the 5 ′ site comprising the first target nucleic acid A second oligonucleotide that is perfectly complementary to the two sites; a second target nucleic acid comprising a first region and a second region, wherein the second region is continuously downstream of the first region And providing a third oligonucleotide wherein at least a portion of the third oligonucleotide is completely complementary to the first site of the second target nucleic acid; at least the site of the first oligonucleotide being Anneal to the first region of the first target nucleic acid and A fourth region comprising a 3 ′ site and a 5 ′ site, wherein at least the 5 ′ site of the rigonucleotide anneals to the second region of the first target nucleic acid and the cleavage of the first cleavage structure is caused by a cleavage factor. Generating a first cleavage structure that produces a fourth oligonucleotide wherein the 5 ′ site is completely complementary to the second site of the second target nucleic acid; At least the site of the nucleotide anneals to the first region of the second target nucleic acid, at least the 5 ′ site of the fourth oligonucleotide anneals to the second region of the second target nucleic acid, and A method is provided that includes generating a second cleavage structure that results in cleavage of the cleavage structure to produce a cleavage fragment; and detecting cleavage of the second cleavage structure. In some embodiments, the 3 ′ site of the fourth oligonucleotide comprises a 3 ′ terminal nucleotide that is not complementary to the second target nucleic acid. In some embodiments, the 3 ′ site of the third oligonucleotide is covalently linked to the second target nucleic acid. In some embodiments, the second target nucleic acid further comprises a 5 ′ region where the 5 ′ region of the second target nucleic acid is a third oligonucleotide. The present invention further includes a cleavage factor; a first oligonucleotide comprising a 5 ′ site complementary to the first site of the target sequence; and a 3 ′ site and a 5 ′ site, wherein the 5 ′ site comprises a target nucleic acid A kit is provided that includes a second oligonucleotide that is complementary to a second site of the target nucleic acid downstream from and contiguous with the first site. In some embodiments, the 3 ′ site of the second oligonucleotide comprises a 3 ′ terminal nucleotide that is not complementary to the target nucleic acid. In a preferred embodiment, the 3 ′ site of the second oligonucleotide comprises a single nucleotide that is not complementary to the target nucleic acid. In some embodiments, the kit further comprises a solid support. For example, in some embodiments, first and / or second oligonucleotides are attached to the solid support. In some embodiments, the kit further comprises a buffer. In some embodiments, the buffer includes a source of divalent cations (eg, Mn ²⁺ ions and / or Mg ²⁺ ions). In some embodiments, the kit further comprises a third oligonucleotide complementary to the third site of the target nucleic acid upstream of the first site of the first target nucleic acid. In yet another embodiment, the kit further comprises a target nucleic acid. In some embodiments, the kit further comprises a second target nucleic acid. In yet another embodiment, the kit further comprises a third oligonucleotide comprising a 5 ′ site complementary to the first region of the second target nucleic acid. In some embodiments, the 3 ′ site of the third oligonucleotide is covalently linked to the second target nucleus. In another specific embodiment, the second target nucleic acid further comprises a 5 'site where the 5' site of the second target nucleic acid is a third oligonucleotide. In yet another embodiment, the kit further comprises an ARRESTOR molecule (eg, an ARRESTOR oligonucleotide).

  The present invention further provides a target nucleic acid comprising a) a first region, a second region, a third region, and a fourth region, wherein the first region is adjacent to the downstream of the second region. A target nucleic acid in which the second region is adjacent downstream of the third region and the third region is adjacent downstream of the fourth region; b) complementary to the fourth region of the target nucleic acid C) a second oligonucleotide comprising a 3 ′ site and a 5 ′ site, wherein the 5 ′ site of the second oligonucleotide is complementary to the second region of the target nucleic acid. A second oligonucleotide wherein the 3 ′ site of the second oligonucleotide comprises a sequence complementary to the third region of the target nucleic acid; and d) a third oligonucleotide comprising a 3 ′ site and a 5 ′ site The 5 ′ site of the third oligonucleotide comprises a sequence complementary to the first region of the target nucleic acid, 3 'site of the oligonucleotide provides a composition comprising a cleavage structure comprising a third oligonucleotide comprising a sequence complementary to a second region of the target nucleic acid.

  The present invention is not limited by the length of the four regions of the target nucleic acid. In one embodiment, the first region of the target nucleic acid is 11-50 nucleotides in length. In another embodiment, the second region of the target nucleic acid is 1-3 nucleotides in length. In another embodiment, the third region of the target nucleic acid is 6 to 9 nucleotides in length. In yet another embodiment, the fourth region of the target nucleic acid is 6 to 50 nucleotides in length.

  The present invention is not limited by the nature or composition of the first, second, third and fourth oligonucleotides, and these oligonucleotides include DNA, RNA, PNA, and combinations thereof It can also contain modified nucleotides, universal bases, adducts and the like. Furthermore, one or more of the first, second, third and fourth oligonucleotides can contain dideoxynucleotides at the 3 ′ end.

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

  As mentioned above, the present invention contemplates the use of structure-specific nucleases in detection methods. In one embodiment, the present invention provides a method for detecting the presence of a target nucleic acid molecule by detecting a non-target cleavage product comprising: a) i) a cleavage means, ii) a first region, a second region, A target nucleic acid comprising a third region and a fourth region, wherein the first region is adjacent downstream of the second region, and the second region is adjacent downstream of the third region The origin of the target nucleic acid in which the third region is adjacent downstream of the fourth region, iii) a first oligonucleotide complementary to the fourth region of the target nucleic acid; iv) the 3 ′ site and the 5 ′ A second oligonucleotide comprising a site, wherein the 5 ′ site of the second oligonucleotide comprises a sequence complementary to the second region of the target nucleic acid, and the 3 ′ site of the second oligonucleotide comprises the target nucleic acid A second oligonucleotide comprising a sequence complementary to the third region; iv) a third comprising a 3 'site and a 5' site An oligonucleotide, wherein the 5 ′ site of the third oligonucleotide comprises a sequence complementary to the first region of the target nucleic acid and the 3 ′ site of the third oligonucleotide is complementary to the second region of the target nucleic acid Providing a third oligonucleotide comprising a specific sequence; b) the first oligonucleotide anneals to the fourth region of the target nucleic acid, and at least the 3 ′ site of the second oligonucleotide anneals to the target nucleic acid. In addition, at least the 5 ′ site of the third oligonucleotide is annealed to the target nucleic acid to create a cleavage structure, which results in cleavage of the cleavage structure, producing non-target cleavage products, each with a 3′-hydroxyl group. Mixing the cleavage means, the target nucleic acid, the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide under such reaction conditions And c) detecting a non-target cleavage product.

  The present invention is not limited by the nature of the target nucleic acid. In one embodiment, the target nucleic acid comprises single stranded DNA. In another embodiment, the target nucleic acid comprises double stranded DNA, and prior to step c), the reaction mixture is treated such that the double stranded DNA is substantially single stranded. In another embodiment, the target nucleic acid comprises RNA and the first and second oligonucleotides comprise DNA.

  The present invention is not limited by the nature of the cutting means. In one embodiment, the cleavage means is a structure-specific nuclease, and a particularly preferred structure-specific nuclease is a thermostable structure-specific nuclease.

  In another preferred embodiment, the thermostable structure-specific nuclease is a chimeric nuclease.

  In an alternative preferred embodiment, the method for detecting non-target cleavage products comprises separating reaction products by electrophoresis and then visualizing the separated non-target cleavage products.

  In another preferred embodiment, one or more of the first, second, and third oligonucleotides comprise a dideoxynucleotide at the 3 ′ end. When using an oligonucleotide comprising a dideoxynucleotide, detection of the non-target cleavage product preferably comprises: a) a non-target labeled with at least one labeled oligonucleotide added to the 3′-hydroxyl group of the non-target cleavage product Incubating the non-target cleavage product with a template-independent polymerase and at least one labeled nucleoside triphosphate under conditions such that a cleavage product is generated, and b) the presence of the labeled non-target cleavage product Detecting. The present invention is not limited by the nature of the template-independent polymerase used, and in one embodiment the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. Is done. When TdT or poly A polymerase is used in the detection step, the second oligonucleotide can include a 5 ′ end label that is a different label than the label present on the labeled nucleoside triphosphate. The present invention is not limited by the nature of the 5 ′ end label, and a variety of suitable 5 ′ end labels are known in the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3. Amidites, Cy5 amidites, and digoxigenin are included.

  In another embodiment, detection of a non-target cleavage product comprises: a) adding at least one oligonucleotide to the 3′-hydroxyl group of the non-target cleavage product to produce a non-target cleavage product with a tail Under such conditions, incubating a non-target cleavage product with a template-independent polymerase and at least one nucleoside triphosphate, and b) detecting the presence of a non-target cleavage product with a tail Process. The present invention is not limited by the nature of the template-independent polymerase used, and in one embodiment the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. Is done. If TdT or poly A polymerase is used in the detection step, the second oligonucleotide can include a 5 ′ end label. The present invention is not limited by the nature of the 5 ′ end label, and a variety of suitable 5 ′ end labels are known in the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3. Amidites, Cy5 amidites, and digoxigenin are included.

In preferred embodiments, the reaction conditions include providing a source of a divalent cation, and particularly preferred divalent cations are Mn ²⁺ ions and Mg ²⁺ ions.

  The present invention further provides a method for detecting the presence of a target nucleic acid molecule by detecting a non-target cleavage product comprising: a) i) a cleavage means, ii) a first region, a second region, and a third A target nucleic acid comprising the region of: wherein the first region is adjacent downstream of the second region and the second region is adjacent downstream of the third region, iii) A first oligonucleotide comprising a 3 ′ site and a 5 ′ site, wherein the 5 ′ site of the first oligonucleotide comprises a sequence complementary to the second region of the target nucleic acid, 'First oligonucleotide containing a sequence complementary to the third region of the target nucleic acid; iv) a second oligo that is 11 to 15 nucleotides in length and contains a 3' site and a 5 'site A nucleotide, wherein the 5 ′ site of the second oligonucleotide is complementary to the first region of the target nucleic acid. Providing a third oligonucleotide comprising a sequence, wherein the 3 ′ site of the second oligonucleotide comprises a sequence complementary to the second region of the target nucleic acid; b) at least the 3 ′ site of the first oligonucleotide Anneals to the target nucleic acid, and at least the 5 ′ site of the second oligonucleotide anneals to the target nucleic acid to create a cleavage structure that results in cleavage of the cleavage structure, each with a 3′-hydroxyl group Mixing the cleavage means, the target nucleic acid, the first oligonucleotide, and the second oligonucleotide under reaction conditions such that a non-target cleavage product is generated, and c) detecting the non-target cleavage product. A method of including is provided. In a preferred embodiment, the cleavage means is a structure specific nuclease, preferably a thermostable structure specific nuclease.

  The present invention is not limited by the length of the various regions of the target nucleic acid. In a preferred embodiment, the second region of the target nucleic acid is 1-5 nucleotides in length. In another preferred embodiment, one or more of the first and second oligonucleotides comprise a dideoxynucleotide at the 3 ′ end. When using an oligonucleotide comprising a dideoxynucleotide, detection of the non-target cleavage product preferably comprises: a) a non-target labeled with at least one labeled oligonucleotide added to the 3′-hydroxyl group of the non-target cleavage product Incubating the non-target cleavage product with a template-independent polymerase and at least one labeled nucleoside triphosphate under conditions such that a cleavage product is generated, and b) the presence of the labeled non-target cleavage product Detecting. The present invention is not limited by the nature of the template-independent polymerase used, and in one embodiment the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. Is done. When TdT or poly A polymerase is used in the detection step, the second oligonucleotide can include a 5 ′ end label that is a different label than the label present on the labeled nucleoside triphosphate. The present invention is not limited by the nature of the 5 ′ end label, and a variety of suitable 5 ′ end labels are known in the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3. Amidites, Cy5 amidites, and digoxigenin are included.

  In another embodiment, detection of a non-target cleavage product comprises: a) adding at least one oligonucleotide to the 3′-hydroxyl group of the non-target cleavage product to produce a non-target cleavage product with a tail Under such conditions, incubating a non-target cleavage product with a template-independent polymerase and at least one nucleoside triphosphate, and b) detecting the presence of a non-target cleavage product with a tail Process. The present invention is not limited by the nature of the template-independent polymerase used, and in one embodiment the template-independent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase (TdT) and poly A polymerase. Is done. If TdT or poly A polymerase is used in the detection step, the second oligonucleotide can include a 5 ′ end label. The present invention is not limited by the nature of the 5 ′ end label, and a variety of suitable 5 ′ end labels are known in the art and include biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3. Amidites, Cy5 amidites, and digoxigenin are included.

  The novel detection method according to the present invention comprises target DNA and RNA, including wild-type and mutant alleles, such as genes from humans or animals, which may or may be related to disease or cancer However, it can be used to detect target DNA and RNA without being limited thereto. Furthermore, the method according to the invention is used to detect and / or identify strains of microorganisms such as bacteria, fungi, protozoa, ciliates, and viruses (especially for detecting and identifying RNA viruses such as HCV). ) Can be used.

  The present invention further provides novel enzymes designed to directly detect, characterize and quantify nucleic acids, particularly RNA. The present invention provides an enzyme that recognizes a specific nucleic acid cleavage structure formed on a target RNA sequence and cleaves this nucleic acid cleavage structure in a site-specific manner to produce a non-target cleavage product. The present invention provides an enzyme with improved ability to specifically cleave the DNA component of a complex comprising DNA and RNA nucleic acid strands.

  For example, the present invention has an altered structure as compared to a native DNA polymerase such that the efficiency in a detection assay that utilizes cleavage of a structure containing a nucleic acid (eg, RNA) is altered (eg, improved). Provide DNA polymerase. In particular, the modified polymerase according to the present invention exhibits improved efficiency in detection assays that utilize cleavage of the DNA component of a cleavage structure (eg, an invasive cleavage structure) containing an RNA target strand.

Increased efficiency in detection assays can be brought about by one or a combination of several improved features. For example, in one embodiment, the enzyme according to the present invention has an improved cleavage rate (k _cat ) of a specific target structure, and thus can produce a large amount of cleavage product within a predetermined time. In another embodiment, the enzyme according to the present invention may have reduced activity or rate of cleaving inappropriate or non-specific structures. For example, in certain aspects of the invention, in certain aspects, one aspect of the improvement is that the difference between the detectable amount of cleavage of a specific structure and the detectable amount of cleavage of another structure is increased. In the point. Thus, the specific target structure of the target structure is compared to the speed of the native enzyme so that the difference between the detectable amount of the cleavage of the specific structure and the detectable amount of the cleavage of another structure is increased. It is an object of the present invention to provide an enzyme in which the difference between the detectable amount of cleavage of a specific structure and the detectable amount of cleavage of another structure is increased due to the reduced cleavage rate. Included in the range. However, the present invention is not limited to enzymes whose differences are improved.

  In a preferred embodiment, the enzyme according to the present invention is a DNA polymerase having a modified synthetic activity and having a modified nuclease activity as described above, compared to the activity of the natural DNA polymerase from which the enzyme is derived. It is particularly preferred that the DNA polymerase is modified so that it not only shows an increase in nuclease activity on the RNA target but also a reduction in the synthesis rate compared to the activity of the natural DNA polymerase. Enzymes with reduced synthesis rates and genes encoding these enzymes have been described (eg, Kaiser et al., J. Biol. Chem., 274: 21387 [1999], Lyamichev et al., Proc. Natl. Acad. Sci. 96: 6143 [1999], US Pat. Nos. 5,541,311, 5,614,402, 5,795,763, and 6,909,606, which are incorporated herein by reference). The present invention contemplates combining modifications such that the resulting 5 ′ nuclease has improved efficiency in RNA detection assays without interfering with synthetic activity.

The present invention contemplates a DNA sequence that encodes a DNA polymerase whose sequence is altered relative to the natural sequence, exhibiting a nuclease activity obtained by modifying the nuclease activity of a natural DNA polymerase. For example, in one embodiment, the DNA sequence encodes an enzyme that has improved the cleavage rate (k _cat ) of a specific target structure such that a large amount of cleavage product is produced within a given time. In another embodiment, the DNA encodes an enzyme with reduced activity or rate that cleaves inappropriate or non-specific structures. In certain embodiments, one aspect of the improvement is that the difference between the detectable amount of cleavage of a specific structure and the detectable amount of cleavage of another structure is increased. Thus, the specific target structure of the target structure is compared to the speed of the native enzyme so that the difference between the detectable amount of the cleavage of the specific structure and the detectable amount of the cleavage of another structure is increased. Providing an enzyme-encoding DNA that reduces the rate of cleavage of a specific structure and the amount of detectable cleavage of another structure will increase due to the reduced rate of cleavage. And within the scope of the present invention. However, the present invention is not limited to polymerases that improve the difference.

  In a preferred embodiment, the DNA sequence encodes a DNA polymerase having the above modified nuclease activity and modified synthetic activity as compared to the activity of the native DNA polymerase from which the improved enzyme was derived. It is particularly preferred that the encoded DNA polymerase has been modified to show not only an increase in nuclease activity on the RNA target but also a decrease in the synthesis rate relative to the activity of the native DNA polymerase.

  The present invention is not intended to be limited by the nature of the modifications necessary to introduce modified nuclease activity. Nor is it intended to be limited by the degree of modification or improvement observed. It is intended that the invention be limited by the polymerase of the altered or unaltered protein, or by the nature of the alteration that alters polymerase synthesis, if the polymerase is also altered to alter synthesis. is not.

The present invention contemplates structure-specific nucleases derived from a variety of biological sources including, but not limited to, mesophilic, chilling, thermophilic, and hyperthermophilic organisms. A preferred structure-specific nuclease is thermostable. Thermostable structure-specific nucleases are produced by the INVADER assay (US Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, and 6,090,543) near the melting temperature (T _m ) of the downstream probe oligonucleotide, and See WO 97/27214 and WO 98/42873, which are incorporated herein by reference) as a result of cleaved probe and non- A cleavage probe is particularly useful in that it allows the target cycle to be turned on and off during the course of the reaction. In one embodiment, the thermostable structure-specific enzyme is Thermus aquaticus, Thermus flavus, Thermus thermophilus, Thermus filiformus, and Thermus scotoductus, A thermostable 5 ′ nuclease selected from the group consisting of modified polymerases derived from a natural polymerase of the Thermus species not limited to: However, the present invention is not limited to the use of thermostable 5 ′ nucleases. For example, certain embodiments of the invention utilize short oligonucleotide probes that can turn target cycling on and off at low temperatures.

  In some embodiments, the present invention is an enzyme that has a heterologous functional domain that provides modified (eg, improved) functionality in a nucleic acid cleavage assay. A composition comprising an enzyme is provided. The present invention is not limited by the nature of the nucleic acid cleavage assay. For example, nucleic acid cleavage assays include assays that cleave nucleic acids directly or indirectly in the presence of enzymes. In certain preferred embodiments, the cleavage assay utilizes a cleavage structure having at least one RNA component. In another particularly preferred embodiment, the cleavage assay utilizes a cleavage structure having at least one RNA component, and the DNA portion of the cleavage structure is cleaved.

  In some preferred embodiments, the enzyme comprises a 5 ′ nuclease or a polymerase. In certain preferred embodiments, the 5 ′ nuclease comprises a thermostable 5 ′ nuclease. In another preferred embodiment, the sequence of the polymerase is modified relative to the native polymerase sequence so that it exhibits a lower DNA synthesis activity than the native polymerase. In certain preferred embodiments, the polymerase is a thermostable polymerase (such as, but not limited to, thermus aquaticus, thermus flavus, thermus thermophilus, thermus filiformis, and thermus scoductactus). Derived polymerase).

  The present invention is not limited by the nature of the modified functionality provided by the heterologous functional domain. Specific examples of modifications include amino acid sequences in which a heterologous functional domain provides improved nuclease activity, improved substrate binding activity, and / or improved background specificity in nucleic acid cleavage assays (eg, There are enzymes, including but not limited to one or more amino acids.

  The present invention is not limited by the nature of the heterologous functional domain. For example, in some embodiments, the heterologous functional domain is two from the polymerase domain of a polymerase (eg, introduced into an enzyme by insertion of a chimeric functional domain, or created by mutation) or Contains more amino acids. In certain preferred embodiments, at least one of the two or more amino acids is derived from the palm or thumb region of the polymerase domain. The present invention is not limited by the identity of the polymerase from which two or more amino acids are selected. In certain preferred embodiments, the polymerase comprises a Thermus thermophilus polymerase. In a particularly preferred embodiment, the two or more amino acids are derived from amino acids 300 to 650 of SEQ ID NO: 1.

  The novel enzymes according to the present invention are wild-type and mutant alleles, such as genes from humans, other animals, or plants, that are or may be related to disease or other symptoms. It can be used to detect target DNA and RNA, including but not limited to genes including target DNA and RNA. Furthermore, the enzyme according to the present invention may be used to detect and / or identify strains of microorganisms such as bacteria, fungi, protozoa, ciliates, and viruses (and in particular hepatitis C virus, human immunodeficiency virus, etc. Can be used to detect and identify viruses with the RNA genome. For example, the present invention is a method of cleaving a nucleic acid comprising the steps of providing an enzyme according to the present invention and a substrate nucleic acid, and the substrate nucleic acid (eg, to generate a cleavage product that can be detected). A method comprising the step of exposing to

からなる群より選択されるアミノ酸配列を有する熱安定性5'ヌクレアーゼを提供する。別の態様において、この5'ヌクレアーゼは、

からなる群より選択されるDNA配列によってコードされる。 In one embodiment, the present invention provides:

A thermostable 5 ′ nuclease having an amino acid sequence selected from the group consisting of: In another embodiment, the 5 ′ nuclease is

Is encoded by a DNA sequence selected from the group consisting of

からなる群より選択される塩基配列である組換えDNAベクターを提供する。好ましい態様において、本発明は、構造特異的ヌクレアーゼをコードする塩基配列であって、

からなる群より選択される塩基配列を有するDNAを含む組換えDNAベクターによって形質転換された宿主細胞を提供する。本発明は、使用する宿主細胞の性質によって制限されることはない。当技術分野では、さまざまな原核生物および真核生物の宿主細胞の中で発現されうる構造特異的ヌクレアーゼをコードする塩基配列を発現させるのに適した発現ベクターがよく知られている。好ましい態様において、宿主細胞は大腸菌（Escherichia coli）細胞である。 The present invention is also a recombinant DNA vector having a base sequence encoding a 5 ′ nuclease, wherein the base sequence is

A recombinant DNA vector having a base sequence selected from the group consisting of: In a preferred embodiment, the present invention is a nucleotide sequence encoding a structure-specific nuclease,

A host cell transformed with a recombinant DNA vector containing a DNA having a base sequence selected from the group consisting of: The present invention is not limited by the nature of the host cell used. Expression vectors suitable for expressing base sequences encoding structure-specific nucleases that can be expressed in a variety of prokaryotic and eukaryotic host cells are well known in the art. In a preferred embodiment, the host cell is an Escherichia coli cell.

  The present invention provides a method for modifying 5 'nucleases relative to native 5' nuclease enzymes so that they exhibit improved efficiency in detection assays that utilize cleavage of RNA-containing structures. . In particular, the modified 5 ′ nuclease produced by the method according to the present invention exhibits improved efficiency in detection methods that utilize cleavage of the DNA portion of a cleavage structure containing an RNA target strand (eg, an invasive cleavage structure). . The improved 5 ′ nuclease resulting from the method according to the invention can be improved by any of the methods discussed herein. An example of a method for evaluating improvements in candidate enzymes is shown.

  For example, the present invention provides a method for producing a modified enzyme having improved functionality in a nucleic acid cleavage assay, the step of providing a test substrate for the enzyme and nucleic acid, and introducing a heterologous functional domain into the enzyme. To produce a modified enzyme, contacting the modified enzyme with a nucleic acid test substrate to produce a cleavage product, and detecting the cleavage product. In some embodiments, the introduction of the heterologous functional domain comprises mutating one or more amino acids of the enzyme. In another embodiment, the introduction of the heterologous functional domain into the enzyme comprises adding a functional domain from one protein (eg, another enzyme) to the enzyme (eg, before adding the functional domain to the protein). Removing a part of the enzyme sequence to replace the functional domain). In a preferred embodiment, the nucleic acid test substrate comprises a cleavage structure. In particularly preferred embodiments, the cleavage structure comprises an RNA target nucleic acid. In yet another preferred embodiment, the cutting structure comprises an invasive cutting structure.

からなる群より選択されるDNAによってコードされている。さらに別の好ましい態様において、キットは、核酸切断産物を検出するための試薬をさらに含む。更に好ましい態様において、核酸切断産物を検出するための試薬は、連続する侵襲的切断反応（sequential invasive cleavage reaction)において使用するオリゴヌクレオチドを含む（例えば、米国特許第5,994,069号を参照されたい）。特に好ましい態様において、連続する侵襲的切断反応において使用する試薬は、蛍光共鳴エネルギー転移（FRET）効果を生じさせる成分で標識されたプローブを含む。 The present invention also provides a nucleic acid treatment kit. One preferred embodiment is a kit comprising a composition comprising one or more improved 5 ′ nucleases. Another preferred embodiment provides a kit comprising a) a composition comprising one or more improved 5 ′ nucleases, and b) an INVADER oligonucleotide and a signal probe oligonucleotide. In some embodiments of the kit according to the present invention, the improved 5 ′ nuclease is derived from a DNA polymerase from a true bacterial species. In a further embodiment, the true bacterial species is thermophilic. In yet a further embodiment, the thermus is a thermophilic organism. In yet a further embodiment, the thermophilic organism is selected from the group consisting of Thermus aquaticus, Thermus flavus, Thermus thermophilus, Thermus filiformis, and Thermus scottodactus. In a preferred embodiment, the improved 5 ′ nuclease is

Is encoded by DNA selected from the group consisting of In yet another preferred embodiment, the kit further comprises a reagent for detecting a nucleic acid cleavage product. In a further preferred embodiment, the reagent for detecting a nucleic acid cleavage product comprises an oligonucleotide for use in a sequential invasive cleavage reaction (see, eg, US Pat. No. 5,994,069). In a particularly preferred embodiment, the reagent used in successive invasive cleavage reactions comprises a probe labeled with a component that produces a fluorescence resonance energy transfer (FRET) effect.

  The present invention also provides a method of treating a nucleic acid comprising: a) providing a first structure-specific nuclease comprising an endonuclease in a solution comprising manganese, and a nucleic acid substrate; b) the substrate substantially comprising Treating the nucleic acid substrate at a high temperature so that it becomes single-stranded, c) reducing the temperature under conditions such that the single-stranded substrate forms one or more cleavage structures, and d) cleaving the cleavage means. A method is provided that includes reacting with the structure to produce one or more cleavage products, and e) detecting the one or more cleavage products. In some embodiments of the method, the endonuclease is a CLEAVASE BN enzyme, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, E. coli Exo III, and a Rad1 / Rad10 complex of Saccharomyces cerevisiae. However, it is not limited to these. In yet another embodiment, the nuclease is a 5 ′ nuclease derived from a thermostable DNA polymerase that exhibits lower activity than the DNA synthesis activity of wild type DNA polymerase, but is identical to the 5 ′ nuclease of wild type DNA polymerase 5 'nuclease that retains the activity of In yet another embodiment, the nucleic acid is selected from the group consisting of RNA and DNA. In a further embodiment, the nucleic acid of step (a) is double stranded.

  The present invention also provides a nucleic acid treatment kit comprising a) a composition comprising at least one purified FEN-1 endonuclease, and b) a solution comprising manganese. In some embodiments of the kit, the purified FEN-1 endonuclease is Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease, Metanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobus fulgidus FEN-1, Sulfolobus solfataricus, Pirobacrum aero Film (Pyrobaculum aerophilum), Thermococcus litoralis, Archaeglobus veneficus, Archaeglobus profundus, Ashidianu・ Bridally (Acidianus brierlyi), Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodicium dictium bro b Thermococcus gorgonarius (Thermococcus gorgonarius), Thermococcus zilligii, Methanopyrus kandleri, Methanocpyrus igneus, Mesanopyrus igneus, Pirococus rik ), And a chimeric FEN-1 endonuclease. In another embodiment, the kit further comprises at least one second structure-specific nuclease. In some embodiments, the second structure-specific nuclease is a 5 ′ nuclease derived from a thermostable DNA polymerase and exhibits a lower activity than the DNA synthesis activity of the wild-type DNA polymerase, but the wild-type DNA polymerase This 5 'nuclease retains the same activity as the 5' nuclease. In yet another embodiment of the kit, a portion of the amino acid sequence of the second nuclease is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase from a Thermus hyperthermic species. In a further embodiment, the hyperthermic species is selected from the group consisting of Thermus aquaticus, Thermus flavus, and Thermus thermophilus. In yet another preferred embodiment, the kit further comprises a reagent for detecting the cleavage product.

  The present invention further includes Sulfolobus solvataricas, Pirobacrum aerofilm, Thermococcus litoralis, Alcaegrobus beneficus, Alkaeglobus profundas, Acidians briellii, Acidians ambivalence, Desulfococcus amylolyticus Endonucleases including, Desulfococcus mobilis, Pyrodictium brochii, Thermococcus gorgonarius, Thermococcus gilligii, Methanopyrus candorelli, Methanococcus igneuus, Pyrococcus horicosii, and Aeropyrum pernicus Provided herein are the compositions, mixtures, methods, and kits described herein. These are compositions containing purified FEN-1 endonucleases of biological origin (specific endonucleases described by the sequences provided herein, as well as variants and homologs), kits containing these compositions , A composition comprising a chimeric endonuclease comprising at least a portion of an endonuclease from these organisms, a kit comprising such a composition, encoding an endonuclease from these organisms (including vectors and host cells) Compositions containing nucleic acids, kits containing such compositions, antibodies against endonucleases, mixtures containing endonucleases derived from these organisms, endonuclease cleavage assays (eg, invasive cleavage assays, CFLP, etc.) And methods including components useful in such methods. Including the door. Examples illustrating the creation, structure, use, and characterization of these endonucleases are provided herein.

  The present invention also provides methods for improving the methods and enzymes disclosed herein. For example, the present invention provides a method for improving an enzyme for any purpose (eg, an amplification reaction, a binding reaction, or other use used in a cleavage reaction), comprising the enzyme disclosed herein, Alter enzymes (eg, change amino acid sequences, add or reduce sequences, add post-translational modifications, add other components, whether or not they are organisms, or other alterations) A method comprising the steps of: Similarly, the present invention is a method for improving the methods disclosed herein, wherein one or more changes are made (eg, changing the composition provided by the method, the order of steps). Also provided are methods that include performing the steps of the method, changing or adding or omitting steps).

Improved efficiency in detection assays results from any of several improved features or combinations thereof. For example, in one embodiment, the enzyme according to the present invention improves the cleavage rate (k _cat ) of a specific target structure so that a large amount of cleavage product is produced within a predetermined time. In another embodiment, the enzyme according to the present invention has a reduced activity or rate of cleaving inappropriate or non-specific structures. For example, in certain aspects of the invention, one aspect of the improvement is that the difference between the detectable amount of cleavage of a specific structure and the detectable amount of cleavage of another structure is increased. Thus, the specific target structure of the target structure is compared to the speed of the native enzyme so that the difference between the detectable amount of the cleavage of the specific structure and the detectable amount of the cleavage of another structure is increased. It is within the scope of the present invention to provide an enzyme that will increase the difference between the detectable amount of cleavage of a specific structure and the detectable amount of cleavage of another structure due to the reduced cleavage rate. included. However, the present invention is not limited to enzymes with improved differences.

  In some preferred embodiments, the present invention is an enzyme having a heterologous functional domain, wherein the heterologous functional domain has a modified (eg, improved) functionality in a nucleic acid cleavage assay. Compositions comprising the provided enzymes are provided. The present invention is not limited by the nature of the nucleic acid cleavage assay. For example, nucleic acid cleavage assays include assays that cleave nucleic acids directly or indirectly in the presence of enzymes. In certain preferred embodiments, the cleavage assay utilizes a cleavage structure having at least one RNA component. In another particularly preferred embodiment, the cleavage assay utilizes a cleavage structure having at least one RNA component, and the DNA portion of the cleavage structure is cleaved.

  The present invention is not limited by the nature of the modified functionality provided by the heterologous functional domain. Specific examples of modifications include amino acid sequences in which a heterologous functional domain provides improved nuclease activity, improved substrate binding activity, and / or improved background specificity in nucleic acid cleavage assays (eg, There are enzymes, including but not limited to one or more amino acids.

  The present invention is not limited by the nature of the heterologous functional domain. For example, in some embodiments, the heterologous functional domain is two from the polymerase domain of a polymerase (eg, introduced into an enzyme by insertion of a chimeric functional domain, or created by mutation) or Contains more amino acids. In certain preferred embodiments, at least one of the two or more amino acids is derived from the palm or thumb region of the polymerase domain. The present invention is not limited by the identity of the polymerase from which two or more amino acids are selected. In certain preferred embodiments, the polymerase comprises a Thermus thermophilus polymerase. In a particularly preferred embodiment, the two or more amino acids are derived from amino acids 300 to 650 of SEQ ID NO: 1.

  The novel enzymes according to the present invention are wild-type and mutant alleles, such as genes from humans, other animals, or plants, that are or may be related to disease or other symptoms. It can be used to detect target DNA and RNA, including but not limited to genes including target DNA and RNA. Furthermore, the enzyme according to the present invention is used to detect and / or identify strains of microorganisms including bacteria, fungi, protozoa, ciliates, and viruses (and in particular hepatitis C virus and human immunodeficiency virus). Can be used to detect and identify viruses with RNA genomes). For example, the present invention is a method of cleaving a nucleic acid comprising the steps of providing an enzyme according to the present invention and a substrate nucleic acid, and the substrate nucleic acid (eg, to generate a cleavage product that can be detected). A method comprising the step of exposing to In some embodiments, the substrate nucleic acid is present in a cell lysate.

  The present invention is also a method for detecting the presence of a target nucleic acid sequence, comprising the steps of cleaving an invasive cleavage structure comprising an RNA target nucleic acid, and detecting that the invasive cleavage structure has been cleaved. I will provide a. Such assays include multiplex assays in which a number of invasive cleavage structures are cleaved. Such structures include structures formed on different target nucleic acids and structures formed at different positions of the target nucleic acid in the sample. In some embodiments, the target nucleic acid comprises a first region and a second region that is continuously downstream from the first region. In some embodiments, the invasive cleavage structure comprises a target nucleic acid, a first oligonucleotide, and a second oligonucleotide, wherein at least a portion of the first oligonucleotide is a first of the first target nucleic acid. And the second oligonucleotide comprises a 3 ′ site and a 5 ′ site, and this 5 ′ site is completely complementary to the second region of the target nucleic acid. In some embodiments, the 3 ′ site of the second oligonucleotide comprises a 3 ′ terminal nucleotide value that is not complementary to the target nucleic acid. In some embodiments, the 3 ′ site of the second oligonucleotide comprises a single nucleotide that is not complementary to the target nucleic acid. In some embodiments, the method further includes forming a second invasive cleavage structure that includes a non-target cleavage product and cleaving the second invasive cleavage structure. In some embodiments, the invasive cleavage structure or the second invasive cleavage structure comprises an oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 709-2640. In another embodiment, the invasive cleavage structure or the second invasive cleavage structure comprises an oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 169-211 and 619-706. In some preferred embodiments, the target nucleic acid comprises cytochrome P450 RNA or cytokine RNA. In some embodiments, the first or second region of the target nucleic acid comprises a splice site, exon (or part thereof), or intron (or part thereof). In some embodiments, the RNA target nucleic acid is provided in a cell lysate. In some embodiments, the first oligonucleotide is covalently linked to the second oligonucleotide. Such oligonucleotides can be used, for example, in the methods described in US Pat. Nos. 5,714,320 and 5,854,033. These documents are incorporated herein by reference in their entirety. The present invention also provides a kit comprising one or more components used in the above method.

A schematic diagram of a continuous invasive cutting reaction is shown. In step A, the upstream INVADER oligonucleotide and the downstream probe bind to the target nucleic acid strand to form a cleavage structure. In step B, a part of the signal probe cleaved from A is combined with the second target nucleic acid strand and the labeled signal probe to form a second cleavage structure. In step C, the labeled second cleavage structure cleaves to produce a detectable signal. The schematic diagram is shown about several examples of the invasive cutting | disconnection structure containing RNA target strand (sequence number: 141). Panel A represents the INVADER oligonucleotide (SEQ ID NO: 142) and probe (SEQ ID NO: 143). Panel B represents the INVADER oligonucleotide (SEQ ID NO: 144) and probe (SEQ ID NO: 143). Panel C represents INVADER oligonucleotide (SEQ ID NO: 145) and probe (SEQ ID NO: 145). Panel D represents the INVADER oligonucleotide (SEQ ID NO: 145) and probe (SEQ ID NO: 146). Two examples of structures that are not labeled invasive cutting structures of SEQ ID NOs: 147-152 are shown in the schematic diagram. 1 shows a schematic diagram of an invasive cleavage structure useful for detecting target sequence mutations. FIG. In A, an invasive cleavage structure with an overlap between the two probes is formed, and the arrows indicate that it can be cleaved with the enzyme of the present invention. In B, the target sequence mutation removes the region complementary to the downstream probe and removes the overlap. If the arrow is not shown in panel B, it indicates that the cutting speed of this structure is lower than that shown in panel A. A diagram of the X-ray structure of the triple complex of primer / template DNA and Klentaq1 in the polymerization method investigated by Li et al. (Li et al., Protein Sci., 7: 1116 [1998]) is shown. Although not showing exact boundaries between physical form features, the parts referred to in the text as “finger”, “thumb” and “palm” regions are roughly represented by circles, squares, and oval shapes, respectively. . The schematic diagram of the DNA polymerase gene derived from Thermus aquaticus is shown. The restriction enzyme sites used in this study are described above. Approximate regions encoding the various structural or functional domains of the protein are indicated by arrows with tails at both ends below. A schematic diagram of a chimeric construct containing TaqPol gene and part of TthPol gene is shown. White boxes and hatched boxes represent TaqPol and TthPol sequences, respectively. The numbers correspond to the amino acid sequence of TaqPol. TaqPol 5 ′ nuclease and polymerase domains are shown, as well as the palm, thumb and finger regions of the polymerase domain. The abbreviations of the restriction enzyme sites used are as follows. E, EcoRI; N, NotI; Bs, BstBI; D, NdeI; B, BamHI; and S, SalI. Comparison of nucleotide structures of polymerase genes isolated from Thermus aquaticus (SEQ ID NO: 153), Thermus flavus (SEQ ID NO: 154) and Thermus thermophilus (SEQ ID NO: 155). Show. A consensus sequence (SEQ ID NO: 156) is shown above each column. FIG. 8B is a diagram showing a continuation of FIG. 8A. FIG. 8C is a diagram showing a continuation of FIG. 8B. FIG. 8D is a diagram showing a continuation of FIG. 8C. FIG. 8E is a diagram showing a continuation of FIG. 8D. FIG. 8F is a diagram showing a continuation of FIG. 8E. FIG. 8G is a diagram showing a continuation of FIG. 8F. FIG. 8H is a diagram showing a continuation of FIG. 8G. A comparison of the amino acid sequences of polymerases isolated from Thermus aquaticus (SEQ ID NO: 157), Thermus flavus (SEQ ID NO: 158) and Thermus thermophilus (SEQ ID NO: 1) is shown. A consensus sequence (SEQ ID NO: 159) is shown above each column. FIG. 9B is a diagram showing a continuation of FIG. 9A. FIG. 9C is a diagram showing a continuation of FIG. 9B. The proposed structure is shown for the substrate sequence of the cleavage signal amplification reaction for the human IL-6 RNA target strand (SEQ ID NO: 160) and upstream probe (SEQ ID NO: 161). The cleavage site of the downstream probe (SEQ ID NO: 162) is indicated by an arrow. The sequence of the IL-6 DNA target strand (SEQ ID NO: 163) is shown below. Image from a fluorograph showing the product of the invasive cleavage assay using the IL-6 substrate shown in Figure 10 with the indicated enzyme and either DNA target strand (A) or RNA target strand (B) Indicates. FIG. 5 shows a comparison of cycling cleavage activity of Taq DN RX HT, Tth DN RX HT and Taq-Tth chimeric enzymes and IL-6 substrate with RNA target strand. A comparison of the amino acid sequences of BstI-BamHI fragments of TaqPol (SEQ ID NO: 164) and TthPol (SEQ ID NO: 165) is shown. Similar amino acid pairs are shaded in dark gray. Parallel amino acids with charge differences are shaded in dark gray. The numbers correspond to the amino acid sequence of TaqPol. Amino acids whose corresponding TthPol amino acids were changed by site-directed mutagenesis were represented by (+). Comparison of cycling cleavage activity of Taq DN RX HT, Taq-Tth chimeric enzyme and chimeric enzyme with the indicated additional amino acid modifications and IL-6 substrate with RNA target strand is shown. Comparison of cycling cleavage activity of Taq DN RX HT, Tth DN RX HT and Taq DN RX HT with the indicated additional amino acid modification and IL-6 substrate with RNA target strand. A comparison of the polymerization activity of TaqPol, TthPol and Taq-Tth chimeric enzymes and TaqPol with the indicated amino acid modifications is shown. A diagram of the X-ray structure of the triple complex of primer / template DNA and Klentaq1 in the polymerization method investigated by Li et al. (Li et al., Protein Sci., 7: 1116 [1998]) is shown. Amino acids G418 and E507 are shown. The schematic diagram of the example of the substrate used for the various cutting activity measurement of an enzyme is shown. The substrate is labeled with fluorescent dyes and quenching groups for FRET detection that facilitates detection and measurement, for example, as indicated. The 18A and 18B substrates are invasive cleavage structures with RNA and DNA target strands, respectively. Both are used for the purpose of evaluating the enzyme activity for another structure in the invasive cleavage reaction. The schematic diagram of the example of the substrate used for the various cutting activity measurement of an enzyme is shown. The substrate is labeled with fluorescent dyes and quenching groups for FRET detection that facilitates detection and measurement, for example, as indicated. The 18A and 18B substrates are invasive cleavage structures with RNA and DNA target strands, respectively. Both are used for the purpose of evaluating the enzyme activity for another structure in the invasive cleavage reaction. The schematic diagram of the example of the substrate used for the various cutting activity measurement of an enzyme is shown. The substrate is labeled with fluorescent dyes and quenching groups for FRET detection that facilitates detection and measurement, for example, as indicated. 18C shows an example of the X structure. Both are used for the purpose of evaluating the enzyme activity for another structure in the invasive cleavage reaction. The schematic diagram of the example of the substrate used for the various cutting activity measurement of an enzyme is shown. The substrate is labeled with fluorescent dyes and quenching groups for FRET detection that facilitates detection and measurement, for example, as indicated. 18D shows an example of a hairpin structure. Both are used for the purpose of evaluating the enzyme activity for another structure in the invasive cleavage reaction. A schematic diagram of a chimeric construct containing TaqPol gene and part of TthPol gene is shown. White boxes and hatched boxes indicate TaqPol and TthPol sequences, respectively. Chimeras also contain DN, RX and HT modifications. The table compares the cleavage activity of each protein against the indicated cleavage substrate. A shows a schematic diagram of RNA containing an invasive cleavage substrate. The 5 ′ end of the target molecule (SEQ ID NO: 166) is modified with biotin as described and blocked with streptavidin. A downstream probe (SEQ ID NO: 167) with a cleavage site is also shown. Panels BD show an analysis of the nature of the Taq DN RX HT G418K / E507Q mutant in cleaving the indicated substrate reacted under conditions of different reaction temperatures, KCl concentrations and MgSO ₄ concentrations. The schematic diagram of the model substrate used for the test of the enzyme regarding invasive cleavage activity is shown. The molecule shown in 21A provides the DNA target strand (SEQ ID NO: 168), and the model shown in 21B provides the RNA (SEQ ID NO: 167) containing the target strand. Both 21A and B represent downstream probes (SEQ ID NO: 166). FIG. 2 shows a schematic diagram of a model substrate used to test enzyme cleavage activity against another non-invasive structure. The schematic diagram of the model substrate used for the test regarding the invasive cleavage activity of an enzyme is shown. The schematic diagram of the model substrate used for the test regarding the invasive cleavage activity with respect to RNA or DNA target strand of an enzyme is shown. A comparison of the cycling cleavage activity of Tth DN RX HT, Taq 2M, TfiPol, Tsc Pol and Tfi and mutant enzymes derived from Tsc is shown. The structure used for the determination of the probe cleavage ability of the enzyme in the presence or absence of the upstream oligonucleotide. FIG. 26 shows oligonucleotide 89-15-1 (SEQ ID NO: 212), oligonucleotide 81-69-5 (SEQ ID NO: 213), oligonucleotide 81-69-4 (SEQ ID NO: 214), oligonucleotide 81- This represents the partial sequence of 69-3 (SEQ ID NO: 215), oligonucleotide 81-69-2 (SEQ ID NO: 216) and M13mpl8 (SEQ ID NO: 217). The image by the fluorescence imaging device which shows upstream duplication oligonucleotide dependence of Pfu FEN-1 regarding the specific cutting | disconnection of a probe is shown. FIG. 28a shows a fluorograph image comparing the amount of product produced in a standard (ie non-continuous invasive cleavage reaction) and a continuous invasive cleavage reaction. FIG. 28b shows the product produced in standard or basic (ie non-continuous invasive cleavage reaction) and continuous invasive cleavage reaction (“INVADER sqrd”) (y axis = fluorescence unit; x axis = attomole target) The graph which compared the quantity of was represented. Fig. 5 shows an image by a fluorescence imaging apparatus showing that the product after the end of a continuous invasive cleavage reaction is not cross-contaminated by a subsequent similar reaction. The oligonucleotide sequence used in the invasive cleavage reaction for detection of HCMV viral DNA is shown. FIG. 30 shows the sequences of oligonucleotides 89-76 (SEQ ID NO: 218), oligonucleotide 89-44 (SEQ ID NO: 219) and nucleotides 3057-3110 (SEQ ID NO: 220) of the HCMV genome. The image by the fluorescence imaging device which shows the highly sensitive detection of HCMV viral DNA in the sample containing human genomic DNA using invasive cleavage reaction is shown. A cleavage probe derived from the first invasive cleavage reaction is utilized as the INVADER oligonucleotide in the second invasive cleavage reaction, and the ARRESTOR oligonucleotide is present at the second probe cleavage with the remaining uncleaved first The schematic diagram of 1 aspect of this invention which inhibits the effect | action of a probe is shown. A cleavage probe derived from the first invasive cleavage reaction is utilized as the integrated INVADER-target complex in the second invasive cleavage reaction, and the ARRESTOR oligonucleotide is left uncut by the second probe cleavage. The schematic diagram of 1 aspect of this invention which inhibits the effect | action of a 1st probe is shown. Three different images of a modified ARRESTOR oligonucleotide with two different lengths of 2′O-methylated and having a 3 ′ terminal amine by a fluorescence imaging apparatus are shown. Both are non-specific for the second probe included in the second step of the reaction utilizing a cleavage probe derived from the first invasive cleavage reaction as an integrated INVADER-target complex in the second invasive cleavage reaction Reduce background cutting. The non-specificity of the cleavage probe derived from the first invasive cleavage reaction increases the concentration of the first probe in the first stage of the reaction utilized as the INVADER oligonucleotide in the second invasive cleavage reaction 2 shows an image from a fluorescence imaging apparatus showing the effect on specific and specific cleavage signals. The concentration of the first probe in the first stage of the reaction is increased and the cleavage probe derived from the first invasive cleavage reaction is used as the INVADER oligonucleotide in the second invasive cleavage reaction. FIG. 6 shows an image from a fluorograph showing the effect on non-specific and specific cleavage signals of including a modified ARRESTOR oligonucleotide that is 2′O-methylated and has a 3 ′ terminal amine in the second step. A cleavage probe derived from the first invasive cleavage reaction is 2'O-methylated in the second step of the reaction utilized as an INVADER oligonucleotide in the second invasive cleavage reaction, and the 3 'terminal amine Microsoft Excel for comparing the effect of increasing the concentration of the first probe in the first step of the reaction on non-specific and specific cleavage signals in the presence or absence of modified ARRESTOR oligonucleotides (MICROSOFT EXCEL) The graph obtained using the software spreadsheet is shown. The non-specification that the cleavage probe derived from the first invasive cleavage reaction comprises an unmodified ARRESTOR oligonucleotide in the second step of the reaction utilized as an INVADER oligonucleotide in the second invasive cleavage reaction And two types of images by a fluorescence imaging apparatus showing the effect on specific cleavage signals. A cleavage probe derived from the first invasive cleavage reaction is used in the second step of the reaction utilized as an INVADER oligonucleotide in the second invasive cleavage reaction, in the 3 ′ terminal amine modified ARRESTOR oligonucleotide, partly 2 Including a modified ARRESTOR oligonucleotide that is' O-methyl substituted and partially has a 3 'terminal amine, or a fully AR'TORylated and completely modified 3' terminal amine Two types of images are shown with a fluorescence imaging device showing the effect on non-specific and specific cleavage signals. The non-existence of the cleavage probe derived from the first invasive cleavage reaction comprising an ARRESTOR oligonucleotide of different length in the second step of the reaction utilized as the INVADER oligonucleotide in the second invasive cleavage reaction. Two images from a fluorescence imaging device are shown comparing the effects on specific and specific cleavage signals. The non-existence of the cleavage probe derived from the first invasive cleavage reaction comprising an ARRESTOR oligonucleotide of different length in the second step of the reaction utilized as the INVADER oligonucleotide in the second invasive cleavage reaction. Two images from a fluorescence imaging device are shown comparing the effects on specific and specific cleavage signals. Here, the longer variant second probe used in the reaction of FIG. 37A is tested. Figure 2 shows a schematic of a first probe juxtaposed with multiple different length ARRESTOR oligonucleotides. The region of the first probe complementary to the HBV target sequence is underlined. The ARRESTOR oligonucleotide was juxtaposed with the probe according to complementarity. A second step in a reaction where a cleavage probe derived from the first invasive cleavage reaction using two different lengths of the second probe is utilized as an INVADER oligonucleotide in the second invasive cleavage reaction Figure 2 shows two images from a fluorograph comparing the effects of different lengths of ARRESTOR oligonucleotides on non-specific and specific cleavage signals. Running average value (Ave ()) calculated for 10 nucleotide strands of human ubiquitin RNA derived from the output of SS count in mfold analysis expressed as a percentage of the total number of structures containing a specific base found by mfold 10) is a graph in which the index) is plotted against the position of the base. An example of a microplate layout for an RNA INVADER assay containing 40 samples, 6 standards, and 1 non-target control is shown. The composition of the INVADER assay used to detect human (h), mouse (m) and rat (r) RNA of the indicated gene or transcript. FIG. 41-2 is a diagram showing a continuation of FIG. 41-1. FIG. 41-3 is a diagram showing a continuation of FIG. 41-2. FIG. 41-4 is a diagram showing a continuation of FIG. 41-3. FIG. 41-5 is a diagram showing a continuation of FIG. 41-4. FIG. 41-6 is a diagram showing a continuation of FIG. 41-5. FIG. 41-7 is a diagram showing a continuation of FIG. 41-6. FIG. 41-8 is a diagram showing a continuation of FIG. 41-7. FIG. 41-9 is a diagram showing a continuation of FIG. 41-8. Shows the computer display of INVADERCREATOR's Order Entry Screen. The computer display of INVADERCREATOR's Multiple SNP Design Selection screen. Shows the computer display of the INVADERCREATOR Designer Worksheet screen. Shows the computer display of the INVADERCREATOR's Output Page screen. Shows the computer display of the INVADERCREATOR Printer Ready Output screen. The component (sequence number: 709-2640) of the INVADER assay used for detection of RNA target nucleic acid is shown. The components are grouped per RNA sample to be detected. If multiple probes, INVADER oligonucleotides, stacker oligonucleotides, ARRESTOR oligonucleotides, or other components are provided, any of the plurality of components can be used unless otherwise indicated. Unless otherwise specified, oligonucleotides are represented in the 5′-3 ′ direction. FIG. 47-2 is a diagram showing a continuation of FIG. 47-1. FIG. 47-3 is a diagram showing a continuation of FIG. 47-2. FIG. 47-4 is a diagram showing a continuation of FIG. 47-3. FIG. 47-5 is a diagram showing a continuation of FIG. 47-4. FIG. 47-6 is a diagram showing a continuation of FIG. 47-5. FIG. 47-7 is a diagram showing a continuation of FIG. 47-6. FIG. 47-8 is a diagram showing a continuation of FIG. 47-7. FIG. 47-9 is a diagram showing a continuation of FIG. 47-8. FIG. 47-10 is a diagram showing a continuation of FIG. 47-9. FIG. 47-11 is a diagram showing a continuation of FIG. 47-10. FIG. 47-12 is a diagram showing a continuation of FIG. 47-11. FIG. 47-13 is a diagram showing a continuation of FIG. 47-12. FIG. 47-14 is a diagram showing a continuation of FIG. 47-13. FIG. 47-15 is a diagram showing a continuation of FIG. 47-14. FIG. 47-16 is a diagram showing a continuation of FIG. 47-15. FIG. 47-17 is a diagram showing a continuation of FIG. 47-16. FIG. 47-18 is a diagram showing a continuation of FIG. 47-17. FIG. 47-19 is a diagram showing a continuation of FIG. 47-18. FIG. 47-20 is a diagram showing a continuation of FIG. 47-19. FIG. 47-21 is a diagram showing a continuation of FIG. 47-20. FIG. 47-22 is a diagram showing a continuation of FIG. 47-21. FIG. 47-23 is a diagram showing a continuation of FIG. 47-22. FIG. 47-24 is a diagram showing a continuation of FIG. 47-23. FIG. 47-25 is a diagram showing a continuation of FIG. 47-24. FIG. 47-26 is a diagram showing a continuation of FIG. 47-25. FIG. 47-27 is a diagram showing a continuation of FIG. 47-26. FIG. 47-28 is a diagram showing a continuation of FIG. 47-27. FIG. 47-29 is a diagram showing a continuation of FIG. 47-28. FIG. 47-30 is a diagram showing a continuation of FIG. 47-29. FIG. 47-31 is a diagram showing a continuation of FIG. 47-30. FIG. 47-32 is a diagram showing a continuation of FIG. 47-31. FIG. 47-33 is a diagram showing a continuation of FIG. 47-32. FIG. 47-34 is a diagram showing a continuation of FIG. 47-33. FIG. 47-35 is a diagram showing a continuation of FIG. 47-34. FIG. 47-36 is a diagram showing a continuation of FIG. 47-35. FIG. 47-37 is a diagram showing a continuation of FIG. 47-36. FIG. 47-38 is a diagram showing a continuation of FIG. 47-37. FIG. 47-39 is a diagram showing a continuation of FIG. 47-38. FIG. 47-40 is a diagram showing a continuation of FIG. 47-39. FIG. 47-41 is a diagram showing a continuation of FIG. 47-40. FIG. 47-42 is a diagram showing a continuation of FIG. 47-41. FIG. 47-43 is a diagram showing a continuation of FIG. 47-42. FIG. 47-44 is a diagram showing a continuation of FIG. 47-43. FIG. 47-45 is a diagram showing a continuation of FIG. 47-44. FIG. 47-46 is a diagram showing a continuation of FIG. 47-45. FIG. 47-47 is a diagram showing a continuation of FIG. 47-46. FIG. 47-48 is a diagram showing a continuation of FIG. 47-47. FIG. 47-49 is a diagram showing a continuation of FIG. 47-48. FIG. 47-50 is a diagram showing a continuation of FIG. 47-49. FIG. 47-51 is a diagram showing a continuation of FIG. 47-50. FIG. 47-52 is a diagram showing a continuation of FIG. 47-51. FIG. 47-53 is a diagram showing a continuation of FIG. 47-52. FIG. 47-54 is a diagram showing a continuation of FIG. 47-53. FIG. 47-55 is a diagram showing a continuation of FIG. 47-54. FIG. 47-56 is a diagram showing a continuation of FIG. 47-55. FIG. 47-57 is a diagram showing a continuation of FIG. 47-56. FIG. 47-58 is a diagram showing a continuation of FIG. 47-57. FIG. 47-59 is a diagram showing a continuation of FIG. 47-58. FIG. 47-60 is a diagram showing a continuation of FIG. 47-59. FIG. 47-61 is a diagram showing a continuation of FIG. 47-60. FIG. 47-62 is a diagram showing a continuation of FIG. 47-61. FIG. 47-63 is a diagram showing a continuation of FIG. 47-62. FIG. 47-64 is a diagram showing a continuation of FIG. 47-63. FIG. 47-65 is a continuation of FIG. 47-64. FIG. 47-66 is a diagram showing a continuation of FIG. 47-65. FIG. 47-67 is a diagram showing a continuation of FIG. 47-66. FIG. 47-68 is a diagram showing a continuation of FIG. 47-67. FIG. 47-69 is a diagram showing a continuation of FIG. 47-68. FIG. 47-70 is a diagram showing a continuation of FIG. 47-69. FIG. 47-71 is a diagram showing a continuation of FIG. 47-70. FIG. 47-72 is a diagram showing a continuation of FIG. 47-71. FIG. 47-73 is a diagram showing a continuation of FIG. 47-72. It is a chart which shows the Ave (10) index with respect to the position of a base pair.

Definitions In order to facilitate understanding of the present invention, several terms and phrases are defined below.

  As used herein, the terms “complementary” or “complementarity” refer to polynucleotides that are related by base pairing rules (ie, sequences of nucleotides such as oligonucleotides or target nucleic acids). For example, the sequence “3′-T-C-A-5 ′” is complementary to the sequence “5′-A-G-T-3 ′”. Complementarity may be “partial” and only some of the bases of the nucleic acid will fit according to the base pairing rules. Alternatively, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids. Both terms may mean each nucleotide, particularly in the context of a polynucleotide. For example, it is noted whether a particular nucleotide in an oligonucleotide is complementary or non-complementary to a nucleotide in another nucleic acid strand as compared to or compared to the complementarity between the remaining oligonucleotide and the nucleic acid strand. Sometimes. With another nucleotide analog (eg, IsoC / IsoG) or with a natural nucleotide (eg, as described in US Pat. No. 5,912,340, which is hereby incorporated by reference in its entirety) Nucleotide analogs used to form unusual base pairings are also considered complementary to the base pairing partner within the meaning of this definition.

  The terms “homology” and “homologous” mean the degree of agreement. There can be partial homology and complete homology. A partially homologous sequence has less than 100% identity with another sequence.

As used herein, the term “hybridization” is used to mean pairing of homologous nucleic acids. Hybridization and the strength of hybridization (ie, the strength of association between nucleic acids) is affected by the degree of complementarity between the nucleic acids, the stringency used, and the T _{m of} the hybrid formed. “Hybridization” methods involve the annealing of one nucleic acid to another complementary nucleic acid, ie, a nucleic acid having a complementary base sequence. It is a well-known phenomenon that two nucleic acid polymers containing complementary sequences recognize each other and anneal by base-pairing interactions. "Hybridization" first discovered by Marmur and Lane, Proc. Natl. Acad. Sci. USA, 46: 453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA, 46: 461 (1960) The phenomenon has since been refined and has become an important tool in modern biology.

  For complementarity, it may be important to determine whether hybridization exhibits complete complementarity or partial complementarity when applied to diagnostics. For example, if you simply want to detect the presence or absence of DNA from pathogenic organisms (eg, viruses, bacteria, fungi, mycoplasma, protozoa), the hybridization method ensures that hybridization occurs when the relevant sequence is present. Only is important and the conditions under which the partially and fully complementary probes hybridize can be selected. However, in other diagnostic applications, it may be necessary to distinguish between partial and complete complementarity by hybridization methods. It may be the purpose to detect genetic polymorphisms. For example, human hemoglobin is partially composed of four polypeptide chains. Two of these chains are the same chain of 141 amino acids (alpha chain) and two are the same chain of 146 amino acids (beta chain). It is known that a gene encoding a beta chain exhibits a polymorphism. The normal allele encodes a beta chain with glutamate at position 6. The mutant allele encodes a beta strand with valine at position 6. This amino acid difference has a significant physiological effect known clinically as sickle cell anemia (most important when the mutant gene is homozygous). The genetic basis of amino acid mutations is related to the single base difference between the sequence of normal gene DNA and that of the mutated gene.

  As used herein, complementation of a nucleic acid sequence means that oligonucleotides “associate in antiparallel” when aligned so that the 5 ′ end of one sequence and the 3 ′ end of the other sequence are aligned. To do. Certain bases not normally present in natural nucleic acids can be included in the nucleic acids according to the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect, and a stable duplex may contain mismatched bases or bases that do not pair at all. Those skilled in the field of nucleic acid technology, for example, empirically consider double-stranded DNA, taking into account variables such as oligonucleotide length, oligonucleotide base composition and sequence, ionic strength, and the incidence of mismatch base pairing. Stability can be determined.

As used herein, the term “T _m ” means “melting temperature”. The melting temperature is the temperature at which half of the population of double-stranded nucleic acid molecules dissociates into single strands. Several equations for calculating the T _m of nucleic acids are known in the art. According to standard references, when nucleic acid is in an aqueous solution of 1 M NaCl, a simple estimate of the T _m value can be calculated by the equation T _m = 81.5 + 0.41 (% G + C). (See Anderson and Young's Quantitative Filter Hybridization section in Nucleic Hybridization (1985)). Other references (eg, Allawi, HT and SantaLucia, J., Jr. “Thermodynamics and NMR of internal GT mismatches in DNA”, Biochemistry 36, 10581-94. (1997)) describes a more sophisticated calculation method that considers not only the characteristics of the sequence but also the structure and surrounding conditions in order to calculate _Tm .

  As used herein, “stringency” means the temperature, ionic strength, and presence of other compounds under conditions for hybridization. Under “high stringency” conditions, nucleic acid base pairing occurs only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, “weak” or “low” stringency is often required when it is desired to hybridize or anneal nucleic acids that are not perfectly complementary to each other.

“High stringency conditions” used for nucleic acid hybridization means 5 × SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O when a probe of about 500 nucleotides in length is used. , And 1.85 g / l EDTA, adjusted to pH 7.4 with NaOH), bind or hybridize at 42 ° C in a solution containing 0.5% SDS, 5x Denhardt's reagent, and 100 µg / ml denatured salmon sperm DNA. It means nucleic acid hybridization including conditions equivalent to soaking and then washing at 42 ° C. in a solution containing 0.1 × SSPE, 1.0% SDS.

The “moderate stringency conditions” used for nucleic acid hybridization are 5 × SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H when using a probe of about 500 nucleotides in length. ₂ O, and 1.85 g / l EDTA, adjusted to pH 7.4 with NaOH), bind or hybridize at 42 ° C in a solution containing 0.5% SDS, 5x Denhardt's reagent, and 100 µg / ml denatured salmon sperm DNA And then nucleic acid hybridization including conditions equivalent to washing at 42 ° C. in a solution containing 1.0 × SSPE and 1.0% SDS.

“Low stringent conditions” means 5 × SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O, and 1.85 g / l EDTA when using a probe of approximately 500 nucleotides in length. , Adjusted to pH 7.4 with NaOH), 0.1% SDS, 5 × Denhardt's reagent [50 × Denhardt's reagent, 500 ml, Ficoll (type 400, Pharmacia) 5 g, BSA (Part V Min; containing 5 g of Sigma), and binding or hybridizing at 42 ° C. in a solution containing 100 g / ml denatured salmon sperm DNA, then in a solution containing 5 × SSPE, 0.1% SDS Means nucleic acid hybridization including conditions equivalent to washing at 42 ° C.

  The term “gene” refers to a DNA sequence that includes control and coding sequences necessary to produce an RNA (eg, ribosomal RNA or transfer RNA), polypeptide, or precursor having a non-coding function. The full-length coding sequence or a portion of the coding sequence can encode RNA or polypeptide as long as the desired activity is retained.

  The term “wild type” refers to a gene or gene product that has the characteristics of a gene or gene product when isolated from a natural source. Wild-type genes are those that are arbitrarily named “normal” or “wild” type genes because they are the most frequently found genes in the population. In contrast, the terms “altered”, “mutated” or “polymorphic” refer to genes that exhibit changes in sequence and / or functional properties (ie, changes in characteristics) compared to wild-type genes or gene products. Or gene product. Note that natural variants can be isolated. That is, these variants are characterized by the fact that their characteristics are altered compared to the wild-type gene or gene product.

  As used herein, the term “recombinant DNA vector” means a DNA sequence containing the desired heterologous sequence. For example, the term is not used solely for sequences that are expressed or that encode an expression product, but in some embodiments, a heterologous sequence is a coding sequence or that is encoded in a host organism. A suitable sequence that is necessary to replicate a sequence or that is required to express a coding sequence that is operably linked in a particular host organism. DNA sequences required for prokaryotic expression include promoters, optionally operator sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

  As used herein, the term “LTR” means a long terminal repeat at each end of a provirus (ie, an integrated retrovirus). The LTR contains a number of regulatory signals such as transcriptional regulators, polyadenylation signals, and sequences required for viral genome replication and integration. The viral LTR is divided into three regions called U3, R, and U5.

  The U3 region contains an enhancer factor and a promoter factor. The U5 region contains a polyadenylation signal. The R (repeat) region separates the U3 region and the U5 region, and a sequence in which the R region is transcribed appears at the 5 ′ end and 3 ′ end of the viral RNA.

  As used herein, the term “oligonucleotide” is two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides, more preferably Is defined as a molecule comprising at least about 15-30 nucleotides. The exact length will depend on many factors, and further on the ultimate function or oligonucleotide usage. Oligonucleotides may be made by any method such as chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.

  Mononucleotides react with the five-membered 5 'phosphate to bind to the 3' oxygen next to it in one direction via a phosphodiester bond, creating an oligonucleotide, 5 'at one end If the phosphate is not attached to the 3 ′ oxygen of the mononucleotide five-membered ring, the end is called the “5 ′ end”, and the oligonucleotide 3 ′ oxygen is followed by the mononucleotide five-membered ring If 5 'phosphate is not bound, it is called "3' end". As used herein, a nucleic acid sequence can be said to have a 5 ′ end and a 3 ′ end, even within long oligonucleotides. The first region on the nucleic acid strand is called upstream of the other region.

  The former when two different non-overlapping oligonucleotides anneal to different regions of one linear complementary nucleic acid sequence and the 3 ′ end of one oligonucleotide points in the direction of the other 5 ′ end Are referred to as “upstream” oligonucleotides, the latter as “downstream” oligonucleotides. Similarly, an oligonucleotide with two overlaps hybridizes to one linear complementary nucleic acid sequence so that the 5 ′ end of the first oligonucleotide is upstream of the 5 ′ end of the second oligonucleotide. When the 3 ′ end of the first oligonucleotide is upstream of the 3 ′ end of the second oligonucleotide, the first oligonucleotide is called the “upstream” oligonucleotide and the second oligonucleotide is “downstream” Called the “side” oligonucleotide.

  The term “primer” means an oligonucleotide that can serve as a starting point for synthesis when placed under conditions under which primer extension is initiated. Oligonucleotide “primers” may be naturally occurring, such as oligonucleotides that have been digested and purified by restriction enzymes, or may be synthetically generated.

  The primer can be selected to be “substantially” complementary to the strand having the particular sequence of the template. The primer must be complementary to the extent that it will hybridize to the template strand to cause primer extension. The primer sequence need not accurately reflect the template sequence. For example, a non-complementary nucleotide fragment may be attached to the 5 ′ end of the primer, and the remaining primer sequence may be substantially complementary to the template strand. Non-complementary bases or longer sequences can be introduced into the primer if the primer sequence is complementary to the template strand so that it can form a complex of template and primer to synthesize primer extension products. Can be scattered.

As used herein, the term “label” means an atom or molecule that can be used to provide a detectable (preferably quantifiable) effect that can bind to a nucleic acid or protein. Labels include dyes; radioactive labels such as ³² P; binding components such as biotin; haptens such as digoxigenin; luminescent, phosphorescent, and fluorogenic components; and fluorescent dyes only, or fluorescence resonance energy transfer ( FRET) may be used in combination with a component that can suppress or shift the emission spectrum, but is not limited thereto. The label can provide a detectable signal by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, and the like. The label may be a charged component (positive or negative charge), or the charge may be neutral. The label may comprise or consist of a nucleic acid or protein sequence as long as the sequence containing the label is detectable.

  As used herein, the term “signal” refers to a detectable effect, such as that caused or provided by a label or assay reaction.

  In this specification, the term “detection device” refers to, for example, an instrument (eg, a camera, a fluorometer, a charge coupled device, a scintillation counter, etc.) or a reaction medium (X-ray or camera film, pH indicator, etc.) Means a device that can communicate the presence of a signal or action to a user or another part of a device (eg, a computer or control device). The detection device is an optical measurement device or a spectroscopic optical measurement device capable of detecting visible light or infrared light such as ultraviolet light, fluorescence or chemiluminescence; a radiation detection device; nuclear magnetic resonance spectroscopy, mass spectrometry, or surface-enhanced Raman spectroscopy. A spectroscopic apparatus of the above; an apparatus such as gel or capillary electrophoresis; or gel exclusion chromatography; or other detection apparatus known in the art, or a combination thereof.

  In this specification, the term “cleavage structure” is a structure formed by the interaction of at least one probe oligonucleotide and a target nucleic acid, and includes a double strand, and the formed structure is cleaved by an enzyme or the like. It means something that can be cut by a factor. A cleavage structure is different from a nucleic acid molecule that is a substrate for nonspecific cleavage by factors such as phosphodiesterase that cleave nucleic acid molecules regardless of secondary structure (ie, formation of a double-stranded structure is not required). A substrate that is specifically cleaved by means.

  In the present specification, the term “folded cleavage structure” means a region of a single-stranded nucleic acid substrate containing a secondary structure, which can be cleaved by an enzymatic cleavage means. A cleavage structure is different from a nucleic acid molecule that is a substrate for nonspecific cleavage by factors such as phosphodiesterase that cleave nucleic acid molecules regardless of secondary structure (ie, formation of a double-stranded structure is not required). A substrate that is specifically cleaved by means.

  As used herein, the term “folded target” refers to at least one secondary structure region (ie, at least one double-stranded region and at least one single-stranded region within a single strand of nucleic acid). Means a nucleic acid strand comprising

  As used herein, the term “cleavage means” or “cleavage factor” is any means capable of cleaving a cleavage structure, including but not limited to enzymes. Examples of the cleavage means include natural DNAP having 5 ′ nuclease activity (eg, Taq DNA polymerase, E. coli DNA polymerase I) and the like. Specifically, it has 5 ′ nuclease activity but does not have synthesis activity. For example, modified DNAP. A “structure-specific nuclease” or “structure-specific enzyme” is an enzyme that recognizes specific secondary structures in nucleic acid molecules and cleaves these structures. The cleaving means according to the present invention cleaves a nucleic acid molecule in response to formation of a cleaving structure, but it is not necessary to cleave a cleaving structure at a special position in the cleaving structure.

  The cutting means is not limited to an enzyme having only 5 ′ nuclease activity. Cleavage means can include nuclease activity provided from a variety of sources, including CLEAVASE enzymes, FEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNA polymerase, and E. coli DNA polymerase I.

  The term “thermostable” refers to an enzyme such as a 5 ′ nuclease and is functional or active (ie, catalyzes) at elevated temperatures, ie, at about 55 ° C. or higher. Can be).

  As used herein, the term “cleavage product” means a product produced by the cleavage means reacting with the cleavage structure (ie, treating the cleavage structure with the cleavage means).

  The term “target nucleic acid” means a molecule comprising a sequence having at least partial complementarity to at least one probe oligonucleotide and at least partial complementarity to an INVADER oligonucleotide. Target nucleic acids can include single-stranded or double-stranded DNA or RNA, and include nucleotide analogs, labels, and other modifiers.

  The term “probe oligonucleotide” means an oligonucleotide that interacts with a target nucleic acid to form a cleavage structure in the presence or absence of an INVADER oligonucleotide. Upon annealing to the target nucleic acid, the probe oligonucleotide and target form a cleavage structure, and cleavage occurs within the probe oligonucleotide.

  The term “non-target cleavage product” means the product of a cleavage reaction that does not occur in the target nucleic acid. As described above, in the method according to the present invention, cleavage of the cleavage structure usually occurs in the probe oligonucleotide. A fragment of the probe oligonucleotide generated by this target nucleic acid-dependent cleavage is a “non-target cleavage product”.

  The term “INVADER oligonucleotide” refers to a portion of an oligonucleotide that hybridizes to a target nucleic acid in the vicinity of a region where the probe and the target nucleic acid hybridize, such as a chimeric moiety or a region that overlaps with the region where the probe and the target hybridize. Means nucleotides, which may or may not be complementary to the target.In some embodiments, the INVADER oligonucleotide is substantially at its 3 'end with a sequence at the 5' end of the probe oligonucleotide. Contains the same sequence.

  With respect to nucleic acid substrates, the term “substantially single-stranded” is in contrast to double-stranded substrates in which the substrate molecule exists primarily as a double-stranded nucleic acid linked by base-pairing interactions between the strands. , Meaning that it exists mainly as a single-stranded nucleic acid.

  As used herein, the term “sequence variation” means a difference in base sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of this wild-type structural gene may change in sequence due to a single base substitution and / or deletion or insertion of one or more nucleotides. is there. These two types of structural genes are said to have different sequences. There may be other variants of the structural gene. This other variant is said to have changed sequence from both the wild type and the first variant.

  As used herein, the term “free” refers to the removal of a nucleic acid fragment from a larger nucleic acid fragment, such as an oligonucleotide, so that the released fragment is no longer covalently bound to the remaining oligonucleotide, for example by the action of 5 nucleases. Means free.

As used herein, the term “K _m ” refers to the Michaelis-Menten constant of an enzyme, and a given enzyme can achieve a rate that is half the maximum rate in an enzyme-catalyzed reaction. Substrate concentration.

  As used herein, the term “nucleotide” includes, but is not limited to, natural nucleotides and / or synthetic oligonucleotides, nucleotide analogs, and nucleotide derivatives. For example, this term includes natural DNA or RNA monomers; peptide nucleic acids (PNA) (M. Egholm et al., Nature 365: 566 [1993]), phosphorothioate DNA, phosphorodithionate DNA, phosphoramidite DNA, amide Nucleotides with modified backbones such as conjugated DNA, MMI-conjugated DNA, 2'-O-methyl RNA, alpha-DNA, and phosphonate DNA; 2'-O-methyl RNA, 2'-fluoro RNA, 2 Nucleotides that are sugar modified, such as' -amino RNA, 2'-O-alkyl DNA, 2'-O-aryl RNA, 2'-O-alkynyl RNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA As well as C-5 substituted pyrimidines (fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazoyl-, imidazolyl-, Substituents such as pyridyl-), fluoro-, 7-deazapurine, inosine with C-7 substituents including lomo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, thiazoyl-, imidazolyl-, pyridyl-, And base-modified nucleotides such as diaminopurine.

  As used herein, the term “base analog” refers to 7-deazapurines (eg, 7-deaza-adenine and 7-deazaguanine); eg, IsoC, IsoG, and all of which are incorporated herein by reference. Other modified bases and nucleotides as described in U.S. Pat.Nos. 5,432,272, 6,001,983, 6,037,120, 6,140,496, 5,912,340, 6,127,121, and 6,143,877 Modified bases that result in altered interactions such as pairings; heterocyclic base analogs based on purine or pyrimidine ring systems, and other heterocyclic bases, such as modified or unnatural forms Means base. Nucleotide analogs include base analogs and include modified forms of deoxyribonucleotides and ribonucleotides.

  The term “polymorphic locus” refers to a locus present in a population that exhibits variation among members of the population (eg, the most common allele frequency is less than 0.95). In contrast, “monomorphic loci” are loci with little or no variation between members of the population (usually the most common allele in the population's gene pool). Are considered loci with a frequency of> 0.95).

  As used herein, the term “microorganism” means an organism that is so small that it cannot be seen with the naked eye, and includes, but is not limited to, bacteria, viruses, protozoa, fungi, and ciliates.

  The term “microorganism gene sequence” refers to a gene sequence derived from a microorganism.

  The term “bacteria” refers to bacterial species, such as true and archaeal species.

  The term “virus” refers to a very small obligate intracellular parasite that cannot replicate autonomously (ie, replication requires host cell machinery).

  The term “multi-drug resistant” refers to a microorganism that is resistant to two or more of the antibiotics or antimicrobial agents used to treat the microorganism.

  The term “sample” in the present specification and claims is used in the broadest sense. On the one hand, it means a specimen or a culture (microorganism culture solution), and on the other hand, it means to include both a biological sample and an environmental sample. Samples can also include synthetic specimens.

  Biological samples are animals, including humans, liquids, solids (eg, stool), or tissues, and liquid and solid foods and feeds such as dairy products, vegetables, meat, meat by-products, and waste Products, and raw materials. Biological samples should be obtained from all of the various families of livestock animals and from wilded or wild animals including but not limited to ungulates, bears, fish, rabbits, rodents, etc. Can do.

  Environmental samples include environmental materials such as surface materials, soil, water, and industrial samples, and samples obtained from food and dairy processing tools, equipment, equipment, cookware, disposable or non-disposable tools, etc. included. These examples should not be construed as limiting the sample types applicable to the present invention.

  The term “origin of target nucleic acid” means a sample containing nucleic acid (RNA or DNA). Particularly preferred target nucleic acid sources include, but are not limited to, blood, saliva, cerebrospinal fluid, pleural effusion, milk, lymph, sputum, and semen.

  If one oligonucleotide is present at a higher concentration than another oligonucleotide (or target nucleic acid), that oligonucleotide is present in an “excess” amount relative to the other oligonucleotide (or target nucleic acid). It is said. When an oligonucleotide, such as a probe oligonucleotide, is present in an excess cleavage reaction relative to the concentration of a complementary target nucleic acid sequence, that reaction is used to determine the amount of target nucleic acid present. be able to. In general, an excess is present because the probe oligonucleotide is present in excess of 100-fold at a molar concentration, typically 1 pmole each if the target nucleic acid sequence is about 10 fmole or less. Will be used.

  Samples “suspected to contain” the first and second target nucleic acids include both those that do and do not contain the target nucleic acid molecule.

  The term “charge-balanced” oligonucleotide means that the modified oligonucleotide carries a charge, and when the modified oligonucleotide is cleaved (ie shortened) or extended, the resulting product becomes Because it has a different charge from the input oligonucleotide (“charge-balanced” oligonucleotide), the charge was modified to allow separation of the input oligonucleotide from the reacted oligonucleotide. Means oligonucleotide. The term “charge balance” does not mean that a modified or balanced oligonucleotide has a net neutral charge (although it may be). Charge balancing means designing and modifying the oligonucleotide to separate specific reaction products resulting from this oligonucleotide input based on the charge from the input oligonucleotide.

という配列（すなわち、修飾塩基を含まない配列番号：136）をもち、プローブの切断が2位と3位の残基の間で起こるINVADERオリゴヌクレオチド特異的切断アッセイ法では、このオリゴヌクレオチドの電荷平衡した配列として考えられるのは、

である。この修飾オリゴヌクレオチドは、正味で負に荷電している。切断後、以下のオリゴヌクレオチドが生成される：

（配列番号：136の3〜22位の残基）。5'Cy3-アミノT-アミノ-T3'は、検出可能な成分（正に荷電したCy3色素）と2個のアミノ修飾塩基をもつ。このアミノ修飾塩基とCy3色素は、リン酸基によって生じる過剰な負の電荷を上回る正の電荷を与えるため、5'Cy3-アミノT-アミノ-T3'は正味の正電荷をもつ。残りの長い方の切断断片は、投入されたプローブと同様、正味の負電荷をもつ。5'Cy3-アミノT-アミノ-T3'断片は、電荷に基づいて、投入プローブ（電荷平衡化オリゴヌクレオチド）から分離できるため、電荷不平衡オリゴヌクレオチドという。長い方の切断産物は、電荷に基づいて、投入プローブ（電荷平衡化オリゴヌクレオチド）から分離することはできない。どちらのオリゴヌクレオチドも正味の負電荷をもつためである。したがって、長い方の切断産物は電荷不平衡オリゴヌクレオチドではない。 For example, the probe oligonucleotide

In the INVADER oligonucleotide specific cleavage assay, where the probe cleavage occurs between residues 2 and 3 (ie, SEQ ID NO: 136 without the modified base), the charge balance of this oligonucleotide The possible arrangement is

It is. This modified oligonucleotide is net and negatively charged. After cleavage, the following oligonucleotides are generated:

(Residues at positions 3-22 of SEQ ID NO: 136). 5'Cy3-amino T-amino-T3 'has a detectable component (positively charged Cy3 dye) and two amino-modified bases. This amino-modified base and Cy3 dye give a positive charge over the excess negative charge caused by the phosphate group, so 5'Cy3-amino T-amino-T3 'has a net positive charge. The remaining longer cut fragment has a net negative charge, similar to the input probe. The 5'Cy3-amino T-amino-T3 'fragment is referred to as a charge unbalanced oligonucleotide because it can be separated from the input probe (charge balanced oligonucleotide) based on charge. The longer cleavage product cannot be separated from the input probe (charge-balanced oligonucleotide) based on charge. This is because both oligonucleotides have a net negative charge. Therefore, the longer cleavage product is not a charge unbalanced oligonucleotide.

  When used with oligonucleotides, such as modified oligonucleotides, the term “net neutral charge” refers to the charge present under the desired reaction or separation conditions (ie, the R—NH 3+ group on thymidine, This shows that the sum of N3 nitrogen and the presence of phosphate groups in cytosine is essentially zero. Oligonucleotides with a net neutral charge do not move the electric field.

  When used with oligonucleotides such as modified oligonucleotides, the term “net positive charge” refers to the charge that exists under the desired reaction or separation conditions (ie, the R—NH 3+ group on thymidine, cytosine). The sum of N3 nitrogen and phosphate groups) is +1 or more. Oligonucleotides with a net positive charge move the electric field towards the cathode.

  When used with oligonucleotides such as modified oligonucleotides, the term “net negative charge” refers to the charge that exists under the desired reaction or separation conditions (ie, the R—NH 3+ group on thymidine, cytosine). The sum of N3 nitrogen and phosphate groups in the case of −1 or less. Oligonucleotides with a net negative charge move the electric field towards the anode.

  The term “polymerization means” or “polymerization factor” means a factor that can facilitate the addition of a nucleoside triphosphate to an oligonucleotide. Preferred polymerization means include DNA polymerase and RNA polymerase.

  The term “ligation means” or “ligation factor” promotes ligation (ie, forming a phosphodiester bond between 3′-OH and 5′P at the ends of two strands of a nucleic acid). It means the factor that can. Suitable ligation means include DNA ligase and RNA ligase.

  In this specification, the term “reactant” has the broadest meaning. The reactive agent is, for example, an enzymatic reactive agent, a chemical reactive agent, or light (for example, ultraviolet light, particularly ultraviolet light having a short wavelength is known to cleave oligonucleotide chains). Agents that can react with the oligonucleotide to shorten (ie, cleave) or extend the oligonucleotide are included within the term “reactant”.

  As used herein, the term “adduct” is used in the broadest sense to mean a compound or factor that can be added to an oligonucleotide. The adduct may be charged (positively or negatively) or the charge may be neutral. Adducts are added to the oligonucleotide either covalently or non-covalently. Examples of adducts include indodicarbocyanine dye amidites, amino substituted nucleotides, ethidium bromide, ethidium homodimers, (1,3-propanediamino) propidium, (diethylenetriamino) propidium, thiazole orange, (N-N ' -Tetramethyl-1,3-propanediamino) propylthiazole orange, (N-N'-tetramethyl-1,2-ethanediamino) propylthiazole orange, thiazole orange-thiazole orange homodimer (TOTO), thiazole orange-thiazole blue Heterodimers (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1), thiazole orange-ethidium heterodimer 2 (TOED2), and fluorescein-ethidium heterodimer (FED), psoralen, biotin, streptavidin , But there is such as avidin, but are not limited to these.

  If the first oligonucleotide is complementary to a region of the target nucleic acid and the second oligonucleotide is complementary to the same region (or part of that region) An area exists. The degree of overlap may vary depending on the nature of complementarity (see, for example, region “X” in FIGS. 29 and 67, and the accompanying discussion).

  As used herein, the term “purified” or “purify” means removing contaminants from a sample. For example, recombinant CLEAVASE nuclease is expressed in bacterial host cells and the nuclease is purified by removing host cell proteins. Thereby, the proportion of such recombinant nucleases is increased in the sample.

  As used herein, the term “recombinant DNA molecule” means a DNA molecule comprising DNA segments joined by molecular biological techniques.

  As used herein, the term “recombinant protein” or “recombinant polypeptide” refers to a protein molecule that is expressed from a recombinant DNA molecule.

  Here, when said about a protein (as “a part of a given protein” etc.), the term “part” means a fragment of that protein. The size of such a fragment is from 4 amino acid residues to 1 minus the total amino acid sequence (eg, 4, 5, 6, ..., (n-1)) .

  As used herein, the term “nucleic acid sequence” refers to oligonucleotides, nucleotides or polynucleotides, and fragments or portions thereof, as well as single or double stranded genomic or synthetic DNA or RNA. Meaning sense strand or antisense strand. Similarly, herein, “amino acid sequence” means a peptide or protein sequence.

  As used herein, the term “peptide nucleic acid” (“PNA”) is a base or base analog that may be present in a natural nucleic acid, but is not a sugar phosphate backbone as in a general nucleic acid. Means a molecule comprising a base or base analog attached to a backbone. Such binding of bases to peptides allows base pairing with complementary nucleobases, similar to oligonucleotides. These small molecules, also named anti-gene factors, bind to the complementary strand of nucleic acids and stop the transcript from growing (Nielsen et al., Anticancer Drug Des. 8: 53-63 [1993]). .

  As used herein, the term "purified" or "substantially purified" refers to a molecule that has been removed from the natural environment and isolated or separated, whether it is a nucleic acid sequence or an amino acid sequence. Means that it is at least 60%, preferably 75%, and most preferably 90% separated from other components that naturally coexist. Thus, an “isolated polynucleotide” or “isolated oligonucleotide” refers to a substantially purified polynucleotide.

  As used herein, the term “fusion protein” refers to a protein of interest (eg, CLEAVASE BN / thrombin nuclease, or a portion thereof) bound to an exogenous protein fragment (a fusion partner comprising a non-CLEAVASE BN / thrombin nuclease protein). ). The fusion partner can enhance the solubility of the recombinant chimeric protein (eg, CLEAVASE BN / thrombin nuclease) when expressed in the host cell, or provide an affinity tag (eg, his-tag) Allows purification of recombinant fusion protein from cells or culture supernatant, or allows both. If desired, the protein of interest (eg, CLEAVASE BN / thrombin nuclease, or a portion thereof) can be removed from the fusion protein by various enzymatic or chemical methods known in the art.

  As used herein, the terms “chimeric protein” and “chimeric protein” mean a single protein comprising amino acid sequence portions derived from two or more types of parent protein molecules. These parent protein molecules may be derived from similar proteins that differ from the origin of the gene, may be different proteins from one organism, or may be different proteins from different organisms. As a non-limiting example, the chimeric structure-specific nuclease according to the present invention is a mixture of amino acid sequences derived from FEN-1 genes derived from two or more organisms having the FEN-1 gene. Can be combined to form a non-natural nuclease. In the present specification, the term “chimera” does not indicate a specific contribution ratio from a natural gene, nor does it limit a method of combining parts of each other. Chimeric structure-specific nuclease constructs with cleavage activity as determined by the test methods described herein are improved cleavage factors that fall within the scope of the present invention.

  As used herein, the term “continuous strand of nucleic acid” refers to a strand of nucleic acid that has a continuous, covalently bonded backbone structure and has no nicks or other disruption moieties. The state of the base portion of each nucleotide, such as whether it is base paired, single stranded or mismatched, is not a requirement for defining a continuous strand. The continuous chain backbone is not limited to the ribose-phosphate or deoxyribose-phosphate composition found in naturally-modified nucleic acids. The nucleic acid according to the present invention includes a phosphorothioate residue, a phosphonate residue, a 2′-substituted ribose residue (for example, 2′-O-methylribose), and another sugar (for example, arabinose) in the structure of the backbone. It may include modifications including but not limited to residues that include.

  As used herein, the term “continuous duplex” refers to a double-stranded nucleic acid region in which the progress of base pairing within the duplex is unbroken (ie, base pairing on the duplex). Is deformed and has no gaps, bumps, or mismatches within a continuous double-stranded region). In this specification, this term only means the way of base pairing in the duplex, and does not imply continuity of the backbone portion of the nucleic acid strand. Double-stranded nucleic acids that are base paired without interruption but nicked on one or both strands are also included in the definition of a continuous double strand.

  The term “double stranded” refers to the state of a nucleic acid in which the base portion of a nucleotide on one strand is bound by a hydrogen bond to a complementary base arranged on the other strand. The condition for becoming double-stranded affects the base state of the nucleic acid. By base pairing, the nucleic acid strand usually has a double stranded tertiary structure with a major and minor groove. Taking a helical state has the potential to be double-stranded.

  The term “double-strand dependent protein binding” refers to the binding of a protein to a nucleic acid, depending on the nucleic acid that is double-stranded or helical.

  As used herein, the term “double-stranded dependent protein binding site or region” means a discrete region or sequence within a nucleic acid to which a double-stranded dependent protein is bound with a particular affinity. . This is quite different from general, non-site-specific double-stranded proteins such as histone proteins that bind to chromatin almost independently of the specific sequence or site.

  As used herein, the term “protein binding region” refers to a nucleic acid region identified by a sequence or structure as being bound to a particular protein or class of proteins. A region that contains genetic information that is identified as such a region when compared to a known sequence, but does not appear to have the structure required for actual binding (eg, a double-stranded nucleic acid binding protein site Single strands) are also included in this definition. In the present specification, the “protein binding region” does not include an endonuclease binding region.

  As used herein, the term “fully double-stranded protein binding region” refers to a continuous double-stranded protein that is required to allow binding or other activity of a double-stranded dependent protein. It means the smallest area. Such a definition is that some double-stranded nucleic acid binding proteins are shorter than the normal protein binding region found in one of the two single strands, but with sufficient activity, This is to include the observation that there is something that can interact. That is, one or more nucleotides in this region can remain unpaired without inhibiting binding. As used herein, the term “fully double-stranded binding region” means the smallest sequence that retains the binding function. Such regions include regions that can accept sequences that are not necessarily double-stranded at multiple positions, but not necessarily simultaneously, so that a single protein-binding region becomes fully protein-binding when double-stranded. There is also a possibility of having a plurality of partial areas.

  The term “template” refers to a nucleic acid strand on which a complementary copy is constructed from a nucleoside triphosphate by the activity of a template-dependent nucleic acid polymerase. Within the duplex, the template is typically illustrated and described as the “lower” strand. Similarly, non-template strands are often illustrated and described as “upper” strands.

  The term “template-dependent RNA polymerase” means a nucleic acid polymerase that produces a new RNA strand by replicating the above-described template strand, but does not synthesize RNA where there is no template. This is opposite to the activity of template-independent nucleic acid polymerases that synthesize or extend nucleic acids without reference to the template, such as terminal deoxynucleotidyl transferase or poly A polymerase.

  The term “ARRESTOR molecule” refers to an agent that is added to or included in an invasive cleavage reaction in order to prevent one or more reaction components from undergoing a subsequent action or reaction. This can be done by sequestering or inactivating any reaction component (eg, by binding or base-pairing a nucleic acid component, or by binding to a protein component). The term “ARRESTOR oligonucleotide” means an oligonucleotide that is included in an invasive cleavage reaction to stop one or more aspects of any reaction (eg, a first reaction, and / or a subsequent reaction or action). However, ARRESTOR oligonucleotides are not limited to specific reactions or reaction steps). This can be done by sequestering any reaction component (eg, base pairing to another nucleic acid or binding to a protein component). However, the term is not limited to the state in which the reaction components are isolated.

  As used herein, the term “kit” refers to a delivery system for delivering a substance. With respect to reaction assays, such delivery systems include reaction reagents (eg, oligonucleotides or enzymes in suitable containers) and / or support materials (eg, buffers, operating instructions for performing the assay). Etc.) means a system that allows storage, movement or delivery from one place to another. For example, the kit includes one or more enclosures (eg, boxes) that contain relevant reaction reagents and / or support materials. As used herein, the term “part kit” means a delivery system that includes two or more separate containers, each containing a portion of the total kit components. These containers are for sending together or separately to the recipient. For example, the first container can contain the enzyme used in the assay, and the second container can contain the oligonucleotide. The term “partial kit” encompasses kits that contain an analyte-specific reagent (ASR) that is regulated by section 520 (e) of the Federal Food, Drug, and Cosmetic Act. However, it is not limited to these. Indeed, a delivery system comprising two or more separate containers, each containing a portion of the total kit component, is also included in the term “part kit”. In contrast, a “combination kit” is a delivery system in which all the components of a reaction assay are contained in a single container (eg, a box with each desired component). The term “kit” includes both partial kits and combined kits.

  As used herein, the term “functional domain” is a region of a protein, or a portion of a region, that provides a functional characteristic of a protein (eg, an enzyme). For example, the functional domain of an enzyme can directly or indirectly provide one or more types of enzyme activity, including but not limited to substrate binding ability and catalytic activity. A functional domain can be characterized by mutating one or more amino acids within the functional domain, i.e., the associated functionality is altered by an amino acid mutation, indicating the presence of the functional domain.

  As used herein, the term “heterologous functional domain” means a functional domain of a protein that is not in its original environment. For example, a heterologous functional domain includes the functional domain of one enzyme introduced into another enzyme. A heterologous functional domain also includes the native functional domain of a protein that has been modified by some method (eg, mutated, added to multiple copies, etc.). A heterologous functional domain can comprise a plurality of contiguous amino acids, or two or more separated amino acids that are amino acid fragments (eg, two or more having non-heterologous intervening sequences). Amino acids or fragments). A heterologous functional domain binds a heterologous amino acid to an amino acid sequence that is not found to be associated with a naturally occurring amino acid sequence in nature, or is associated with a portion of a protein that does not occur in nature. Can be distinguished from the endogenous functional domain.

  As used herein, the term “change in functionality in a nucleic acid cleavage assay” is a characteristic of an enzyme that is altered in some way and differs from its natural state (eg Means different) features. Modifications include, but are not limited to, addition of heterologous functional domains (eg, mutation, or creation of chimeric proteins). In some embodiments, the characteristics of the modified enzyme may be that improved enzyme efficiency in a nucleic acid cleavage assay. Types of improvement are specific to possessing improved nuclease activity (eg, increased reaction rate), improved substrate binding (eg, higher specificity, higher substrate turnover, etc.) Such as enhancing or reducing the binding of certain nucleic acid species [eg, RNA or DNA], and improving background specificity (eg, reducing the production of undesirable products), etc. It is not limited. The present invention is not limited by the nucleic acid cleavage assay used to examine improved functionality. However, in some embodiments of the invention, invasive cleavage assays can be used as nucleic acid cleavage assays. In certain specific embodiments, invasive cleavage assays that utilize RNA targets can be used as nucleic acid cleavage assays.

  Here, the terms “N-terminal” and “C-terminal” with respect to a polypeptide sequence mean a polypeptide region comprising a portion of the N-terminal region and C-terminal region of the polypeptide, respectively. The sequence including the N-terminal region of the polypeptide is a sequence mainly including the N-terminal half of the polypeptide chain, but is not limited to such a sequence. For example, the N-terminal sequence can also include an internal site of a polypeptide sequence that includes bases that include both the N-terminal or C-terminal half of the polypeptide. The same is true for the C-terminal region. The N-terminal region and the C-terminal region can include, but need not include, the amino acids that define the N-terminal and C-terminal extreme ends of the polypeptide, respectively.

DESCRIPTION OF THE INVENTION Introduction The present invention relates to methods and compositions for processing nucleic acids, and in particular to methods and compositions for detecting and characterizing nucleic acid sequences and mutations in the sequences.

  In a preferred embodiment, the present invention relates to a method for nucleic acid cleavage structure in a site-specific manner. The present invention provides various cleavage factors, and in some embodiments, the present invention relates to cleavage enzymes with 5 ′ nuclease activity without interfering with nucleic acid synthesis activity. In another aspect, the present invention provides novel polymerases (eg, thermostable polymerases) having modified polymerase and / or nucleotide activity.

  For example, in some embodiments, the present invention provides a 5 ′ nuclease derived from a thermostable DNA polymerase that exhibits altered DNA synthesis activity that differs from the DNA synthesis activity of natural thermostable DNA polymerase. The 5 'nuclease activity of this polymerase is maintained even if synthetic activity is reduced or lost. Such 5 ′ nucleases can catalyze structure-specific cleavage of nucleic acids in the absence of interfering synthetic activity. If there is no synthetic activity during the cleavage reaction, a nucleic acid cleavage product of uniform size is obtained.

  The novel properties of the nuclease according to the present invention form the basis of a method for detecting specific nucleic acid sequences. This method does not depend on amplifying the target sequence itself as in the current specific target sequence detection method, but on the amplification of the detection molecule.

  DNA polymerase (DNAP) is an enzyme that is isolated from E. coli, thermophilic bacteria of the genus Thermus, and other organisms to synthesize new DNA strands. Some of the known DNAPs contain related nuclease activity in addition to the enzyme synthesis activity.

  DNAP is known to remove nucleotides from the 5 ′ and 3 ′ ends of DNA strands (Kornberg, DNA Replication, WH Freeman and Co., San Francisco, pp. 127-139 [1980 ]). These nuclease activities are usually referred to as 5 'exonuclease and 3' exonuclease, respectively. For example, 5 'exonuclease activity located in the N-terminal domain of some DNAPs removes RNA primers during lagging strand synthesis during DNA replication or removes damaged nucleotides during repair Involved in. Some DNAPs, such as E. coli DNA polymerase (DNAPEcI), also have 3 ′ exonuclease activity that is involved in proofreading during the DNA synthesis process (Kornberg, supra).

  DNAP isolated from Thermus aquaticus, named Taq DNA polymerase (DNAP Taq), has a 5 'exonuclease but no functional 3' exonucleolytic domain (Tindall and Kunkell, Biochem ., 27: 6008 [1988]). Derivatives of DNAPEcI and DNAP Taq are referred to as Klenow and Stoffel fragments, respectively, and lack the 5 ′ exonuclease domain as a result of enzymatic or genetic manipulation (Brutlag et al., Biochem. Biophys. Res. Commun. 37: 982 [1969]; Erlich et al., Science 252: 1643 [1991]; Setlow and Kornberg, J. Biol. Chem., 247: 232 [1972]).

  It has been reported that the 5 'exonuclease activity of DNAP Taq requires simultaneous synthesis (Gelfand, "PCR Technology- Principles and Applications for DNA Amplification") HA Erlich, [ed.], Stockton Press, New York, p. 19 [1989]). The degradation products of DNAPEcI and DNAP Taq by 5 ′ exonuclease are mostly mononucleotides, but short oligonucleotides (≦ 12 nucleotides) are also observed. This suggests that these so-called 5 'exonucleases can function endonucleolytically (Setlow supra; Holland et al., Proc. Natl. Acad. Sci. USA 88: 7276 [1991]).

  In WO 92/06200, Gelfand et al. Reveal that a suitable substrate for the 5 ′ exonuclease activity of thermostable DNA polymerase is a substituted single-stranded DNA. Hydrolysis of the phosphodiester bond occurs between the displaced single stranded DNA and the double helical DNA, with the preferred exonuclease cleavage site being the phosphodiester bond in the double helix region. Thus, this 5 ′ exonuclease activity, usually associated with DNAP, is a structure-dependent single-stranded endonuclease and more precisely called a 5 ′ nuclease. An exonuclease is an enzyme that cleaves a nucleotide molecule from the end of a nucleic acid molecule. On the other hand, an endonuclease is an enzyme that cleaves a nucleic acid molecule inside rather than at a terminal site. Some thermostable DNA polymerase nuclease activities result in endonuclease-like cleavage, which requires contact with the 5 ′ end of the molecule to be cleaved. These nucleases are therefore referred to as 5 'nucleases.

  When 5 'nuclease activity is associated with eubacterial type A DNA polymerase, it has been found that there is an independently functioning domain in the N-terminal third region of this protein. The two-thirds region of the C-terminal side of this molecule constitutes a polymerization domain involved in DNA synthesis. Some type A DNA polymerases have the 3 ′ exonuclease activity associated with the two thirds of the C-terminal side of the molecule.

  The 5 'exonuclease activity and polymerization activity of DNAP can be separated by proteolytic cleavage or genetic manipulation of the polymerase molecule. The Klenow fragment or proteolytic large fragment of DNAPEcI contains polymerase activity and 3 ′ exonuclease activity but does not have 5 ′ nuclease activity. The Stoffel fragment of DNAP Taq (DNAPStf) does not have a 5 'nuclease by genetic manipulation to delete the N-terminal 289 amino acids of the polymerase molecule (Erlich et al., Science 252: 1643 [1991]). WO 92/06200 discloses a thermostable DNAP with a modified 5 ′ to 3 ′ nuclease. US Pat. No. 5,108,892 discloses a Thermus aquaticus DNAP without a 5 ′ to 3 ′ nuclease. Thermostable DNA polymerases with reduced amounts of synthetic activity are available (Third Wave Technologies, Madison, Wis.), US Pat. Nos. 5,541,311, 5,614,402, 5,795,763 No. 5,691,142 and 5,837,450. These documents are incorporated herein by reference in their entirety. The present invention provides a 5 ′ nuclease derived from a thermostable type A DNA polymerase, which retains 5 ′ nuclease activity but has reduced or no synthetic activity. . The ability to decouple enzyme synthesis activity from 5 'nuclease activity proves that 5' nuclease activity does not require DNA synthesis to occur simultaneously as previously reported. (Gelfand, PCR technology, supra).

  In addition to the 5 ′ exonuclease domain of the eubacterial DNA polymerase I protein described above, 5 ′ nucleases have also been found to be involved in bacteriophages, eukaryotes, and archaea. Overall, all of this family of enzymes show very similar substrate specificity despite a limited degree of sequence similarity. As a result, enzymes suitable for use in the method according to the invention can be isolated or derived from a variety of sources.

  A mammalian enzyme with functional similarity to the 5 ′ exonuclease domain of E. coli Pol I was isolated about 30 years ago (Lindahl et al., Proc. Natl. Acad. Sci. USA, 62 (2 ): 597-603 [1969]). Later, another member of this group of enzymes, called the flap endonuclease (FEN1) from eukaryotes and archaea, has almost the same structure-specific activity despite limited sequence similarity. (Harrington and Lieber, Embo J 13 (5), 1235-48 [1994]; Murante et al., J. Bio. Chem 269 (2), 1191-6 [1994]; Robins et al., J Bio. Chem 269 (46), 28535-8 [1994]; Hosfield et al., J. Bio. Chem 273 (42), 27154-61 [1998]). Examination of the substrate specificity of the FEN1 enzyme and the enzymes of eubacteria and related bacteriophages revealed that all enzymes were similar (Lyamichev et al., Science 260 (5109), 778-83 [1993] Harrington and Lieber, supra; Murante et al., Supra; Hosfield, supra; Rao et al., J. Bacteriol. 180 (20), 5406-12 [1998] ;, Bhagwat et al., J. Bio. Chem 272 (45), 28523 -30 [1997]; Garforth and Sayers, Nucleic Acids Res. 25 (19), 3801-7 [1997]).

  Using a shaped substrate, many of the studies cited above have determined that these nucleases leave gaps upon cleavage, so the authors assume that the polymerase fills the gap and can be ligated. It came to think that it should make. Numerous other 5 'nucleases have been shown to leave gaps or duplications after cleavage of the same or similar flap substrate. Since then, structure-specific 5'-exonucleases are thought to leave nicks after cleavage if there is an overlap between the upstream and downstream duplexes of the substrate (Kaiser et al., J. Biol. Chem. 274 (30): 21387-21394 [1999]). Double strands with overlapping bases of several bases can adopt various conformations by branch point migration, but the last nucleotide at the 3 'end of the upstream strand is an unpaired conformation In addition, it was considered that the cleavage rate was essentially the same regardless of whether the end of the upstream primer was A, C, G, or T. It was thought that it was the positional overlap between the 3 ′ end of the upstream primer and the downstream duplex, rather than the sequence overlap, that resulted in optimal cleavage. In addition to leaving these enzymes nicked after cleavage, a single base of the overlap causes cleavage by the enzyme several orders of magnitude faster than when the substrate has no overlap (Kaiser et al., Supra).

  The following 5 ′ nucleases can be applied to some embodiments of the methods described herein. Specific examples of 5 ′ nucleases that can be used in the method according to the present invention include polymerases derived from thermus species including thermus aquaticus, thermus flavus, thermus thermophilus, thermus filiformis, and thermus scottodactus, and modifications. There are, but are not limited to, polymerases. In particular, modified polymerases that exhibit improved efficiency in detection assays that utilize cleavage of DNA moieties with invasive cleavage structures that contain RNA target strands are preferred.

  Methanococcus jannasii, methanobacterium thermoautotrophic cam, alkaegrobus beneficus, sulpholobus solfatalicas, pyrobacrum aerofilm, thermococcus litoralis, alkaecrobus profundas, asidianus briberlii, assidian ambivalence Desulfococcus amylolyticus, Desulfococcus mobilis, Pyrodictium brochii, Thermococcus gilligii, Thermococcus gorgonarius, Methanopiras candreri, Methanococcus igneuus, Pyrococcus horicosii, and Aeropirum A chimeric polymer containing one or more FEN nuclease sites, but not limited to, a Pernicus FEN nuclease. Over zero will be applied, but particularly preferred FEN1 enzymes are Arukaegurobusu fulgidus and Pyrococcus furiosus chimeric. Particularly preferred are modified polymerases that exhibit improved efficiency in detection assays that utilize cleavage of DNA moieties with invasive cleavage structures that contain RNA target strands.

A detailed description of the invention is disclosed in the following sections.
I. Detection of specific nucleic acid sequences using 5 'nucleases in INVADER-specific cleavage assays
II. Signal enhancement by incorporating products of invasive cleavage reactions into successive invasive cleavage reactions
III. Effect of ARRESTOR oligonucleotides on signal and background in a continuous invasive cleavage reaction
IV. Improved enzyme for use in INVADER oligonucleotide-specific cleavage reactions involving RNA targets
V. Reaction design for detection of RNA targets by INVADER assay
VI. A kit for performing the RNA INVADER assay, and
VII. INVADER assay for direct detection and measurement of specific RNA analytes

I. Detection of specific nucleic acid sequences using 5 'nucleases in INVADER-specific cleavage assays
1． INVADER Assay Reaction Design The present invention provides a method for cleaving a nucleic acid cleavage structure to form a nucleic acid cleavage structure determined by the presence of a target nucleic acid and release a characteristic cleavage product. For example, when a target-dependent cleavage structure is cleaved using 5 ′ nuclease activity, the resulting cleavage product is an indication that a specific target nucleic acid sequence is present in the sample. Invasive cleavage can occur when two strands of nucleic acid or both strands of an oligonucleotide hybridize to a target nucleic acid strand to form an overlapping invasive cleavage structure, as described below. Interaction of a cleaving factor (eg, 5 'nuclease) with an upstream oligonucleotide can cause the cleaving factor to cleave the downstream oligonucleotide at an internal site in such a way that a characteristic cleavage product is produced. . Such an embodiment has been named INVADER assay (Third Wave Technologies), and U.S. Patent Applications 5,846,717, 5,985,557, 5,994,069, 6,001,567 and 6,090,543, and It is described in International Publication No. 97/27214 and International Publication No. 98/42873. These documents are incorporated herein by reference in their entirety.

  The present invention further eliminates the need to use temperature cycling (ie, to periodically denature target nucleic acid strands) or to synthesize nucleic acids (ie, substitution utilizing polymerization of target or probe nucleic acid strands). Provided is an assay that reuses or reuses a target nucleic acid while performing multiple hybridization with an oligonucleotide probe and cleaving the probe. When the cleavage reaction is performed under the condition that the probe is continuously displaced on the target strand (ie, by probe-probe displacement or by the equilibrium between probe / target association and dissociation, or a combination involving these mechanisms) [Reybnaldo et al., J. Mol. Biol. 97: 511 (2000)]), multiple probes can hybridize to one target, allowing multiple cleavages and production of multiple cleavage products.

  Depending on the degree of complementarity to the target nucleic acid strand, it can be said that the oligonucleotide defines a specific region of the target. In an invasive cleavage structure, the two oligonucleotides define or hybridize to adjacent target regions (ie, regions that do not have extra target sequence between them). Either or both oligonucleotides can contain extra sites that are not complementary to the target strand. In addition to hybridizing adjacent, in order to form an invasive cleavage structure, the 3 ′ end of the upstream oligonucleotide must contain an additional portion. When both oligonucleotides hybridize to the target strand to form a structure and such 3 ′ end portion is present in that structure on the upstream oligonucleotide, it can be said that the oligonucleotides overlap, This structure can be referred to as an overlapping or invasive cutting structure.

  In one embodiment, the 3 ′ portion of the invasive cleavage structure is a single nucleotide. In this embodiment, the 3 ′ portion can be any nucleotide (ie, it can be complementary to the target strand, but need not be). In a preferred embodiment, the 3 ′ moiety is a single nucleotide that is complementary to the target strand. In another embodiment, the 3 ′ moiety is a nucleotide-like compound (ie, a nucleotide analog, or a moiety having chemical properties similar to nucleotides, such as an organic cyclic compound; see, eg, US Pat. No. 5,985,557) . In yet another embodiment, the 3 ′ moiety is one or more nucleotides that in sequence replicate one or more nucleotides present at the 5 ′ end of the hybridizing region of the downstream oligonucleotide. In a further embodiment, the sequence that replicated the 3 ′ portion of the nucleotide is followed by a single nucleotide that is another nucleotide, rather than a further replica of the downstream oligonucleotide sequence. In yet another embodiment, there is a nucleotide-like compound as described above after the replicated sequence of nucleotides at the 3 ′ portion.

  The downstream oligonucleotide can have, but need not, have additional moieties attached to both ends of the region that hybridizes to the target nucleic acid strand. In a preferred embodiment, the downstream oligonucleotide comprises a portion at its 5 ′ end (ie, a 5 ′ portion). In particularly preferred embodiments, the 5 'moiety is a 5' flap or arm comprising a base sequence that is not complementary to the target nucleic acid strand.

  When an overlapping cleavage structure is formed, a nuclease specific to this structure (ie, by recognizing this structure rather than recognizing the base sequence of the nucleic acid that forms this structure, one of the overlapping structures) Alternatively, it is recognized and cleaved by a nuclease that cleaves a plurality of nucleic acids. Such nucleases are referred to as “structure-specific nucleases”. In some embodiments, the structure-specific nuclease is a 5 ′ nuclease. In a preferred embodiment, the structure-specific nuclease is a 5 ′ nuclease of DNA polymerase. In another preferred embodiment, the DNA polymerase with the 5 ′ nuclease is not capable of synthesis. In another preferred embodiment, the 5 ′ nuclease is a FEN-1 endonuclease. In particularly preferred embodiments, the 5 ′ nuclease is thermostable.

  In some embodiments, the structure-specific nuclease selectively cleaves downstream oligonucleotides. In a preferred embodiment, the downstream oligonucleotide is cleaved at the 5 ′ terminal nucleotide of the region hybridizing to the target in the overlapping structure. When the overlapping structure is cleaved at any position by the structure-specific nuclease, a part or fragment of the nucleic acid is released, and this is called “cleavage product”.

  In some embodiments, cleavage of the overlapping structure is performed under conditions where one or more nucleic acids within the structure dissociate (ie, dehybridize or melt) from the structure. In one embodiment, the dissociation of all or part of a cleavage structure allows the target nucleic acid to form yet another one or more overlapping cleavage structures. In a preferred embodiment, this initial cleavage structure is partially dissociated. In a particularly preferred embodiment, only the cleaved oligonucleotide is dissociated from the original cleavage structure and replaced by another copy of the same oligonucleotide. In some embodiments, the dissociation is induced by increasing the temperature such that no more than one oligonucleotide can hybridize to the target. In another embodiment, the dissociation occurs by cleaving the oligonucleotide to produce only cleavage products that cannot bind to the target strand under the reaction conditions. In preferred embodiments, the conditions are such that the nucleotide associates (ie hybridizes) with the target strand or can be dissociated from the target strand with or without cleavage, and the oligonucleotide hybridizes to the target as part of the overlapping portion of the cleavage structure. Select the conditions for cutting when soyed. In certain preferred embodiments, the number of copies of the oligonucleotide that is cleaved when it becomes part of the overlapping structure portion exceeds the copy number of the target nucleic acid strand, and the target strand is Conditions are selected that are sufficient to be more likely to associate with the original copy of the oligonucleotide than to associate with the cleaved copy of the oligonucleotide.

  In some embodiments, the cleavage is performed by a structure-specific nuclease that can recognize and cleave non-overlapping structures. In a preferred embodiment, the cleavage is performed by a structure-specific nuclease that has a lower rate of cleavage of non-overlapping nucleic acid structures than the rate of cleavage of overlapping structures. In a particularly preferred embodiment, the cleavage is carried out with a structure-specific nuclease whose cleavage rate of non-overlapping nucleic acid structures is slower than 1% compared to the rate of cleavage of overlapping structures.

  In some embodiments, it is desirable to detect the cleavage of overlapping cleavage structures. Detection can be performed by analysis of cleavage products or analysis of remaining uncleaved nucleic acids. For convenience, the analysis of cleavage products is described below, but those skilled in the art will appreciate that these methods can be readily applied to the analysis of non-cleavable nucleic acids in an invasive cleavage reaction. Methods known in the art for analyzing nucleic acids, nucleic acid fragments or oligonucleotides can be applied to the detection of cleavage products.

  In one embodiment, cleavage products can be identified by the chemical content contained therein, for example, each atom, a particular type of reactive group, or each base of a nucleotide (Chargaff et al., J. Biol Chem. 177: 405 [1949]). In this way, it becomes possible to distinguish the cleavage product from a long nucleic acid that liberates it by cleavage, or from another nucleic acid.

  In another aspect, cleavage products can be distinguished by specific physical attributes such as, but not limited to, length, mass, charge, or charge to mass ratio. In yet another embodiment, the cleavage product is a rotational speed in solution, a migration speed by electrophoresis, a sedimentation speed in centrifugation, a time of flight in MALDI-TOF mass spectrometry, a migration speed or other behavior in chromatography, complementary It can be distinguished by behavior related to physical attributes such as, but not limited to, melting temperature from typical nucleic acids, or precipitation potential from solution.

Cleavage product can be detected by release of the label. Such labels include one or more dyes, radioactive labels such as ³² P or ³⁵ S, binding components such as biotin, mass tags such as metal ions or chemical groups, charge tags such as polyamines or charged dyes, digoxigenin, etc. Haptens, luminescent, phosphorescent, and fluorogenic components, and components that can suppress or shift the emission spectrum by fluorescent dyes alone, or by fluorescence resonance energy transfer (FRET), or collisional fluorescence energy transfer, etc. Although there are fluorescent dyes used in combination, it is not limited to these.

  In some embodiments, analysis of cleavage products includes physical analysis and separation by, for example, electrophoresis, selective hybridization or hybridization to a support, or mass spectrometry such as MALDI-TOF. In another embodiment, the analysis detects a change in fluorescence induced by cleavage, as in a FRET-based analysis method, or a change in fluorescence induced by cleavage of nucleic acid rotation rate in solution in fluorescence polarization analysis. It can be carried out without physical analysis or separation methods.

  The cleavage product can then be used in reactions or readout methods that can utilize oligonucleotides. Such reactions include, but are not limited to, modification reactions such as ligation, tail addition by template-independent nucleic acid polymerase, and primer extension by template-dependent nucleic acid polymerase. The modification of the cleavage product can be done for the purpose of adding one or more labels or binding components, changing the mass, adding specific sequences, etc., or to facilitate analysis of the cleavage product, or another byproduct, cleavage reaction This can be done for other purposes such as facilitating analysis of the results.

  Analysis of the cleavage product can also include subsequent steps or reactions that do not modify the cleavage product itself. For example, the cleavage product can be used to complete a functional structure such as a promoter capable of performing transcription in vivo, or another protein binding site. The analysis includes using the completed functional structure or performing the function. One or more cleavage products can be used to complete an overlapping cleavage structure, thereby detecting the product, or the product being involved in further cleavage reactions, etc. Subsequent cleavage reactions used by the method are possible.

  Certain preferred embodiments of the invasive cleavage reaction are described in the following description. In some embodiments, the methods according to the invention use at least one set of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for a structure-specific nuclease. In some embodiments, the cleavage structure is: i) a single-stranded or double-stranded target nucleic acid (when a double-stranded target nucleic acid is used, it can be made single-stranded, for example, by heat treatment); ii) A first oligonucleotide referred to as a “probe” that defines the region by being complementary to the first region of the target nucleic acid sequence; iii) referred to as an “INVADER oligonucleotide” A second oligonucleotide, whose 5 ′ site is adjacent downstream of the first target region and defines a second region of the same target nucleic acid sequence, the second part comprising It includes a second oligonucleotide that overlaps the region defined by the oligonucleotide.

  The binding of these oligonucleotides in this embodiment is achieved by dividing the target nucleic acid into three separate regions: a region that is complementary only to the probe, a region that is complementary only to the INVADER oligonucleotide, and a region that is complementary to both oligonucleotides. It is thought that it is divided into areas. As noted above, in some aspects of the invention, the overlap can include portions other than overlapping complementary bases. Thus, in some embodiments, there is a physical overlap between the INVADER oligonucleotide and the probe oligonucleotide, but no sequence overlap. That is, in the latter embodiment, there is no target nucleic acid region having complementarity to both oligonucleotides.

a) Oligonucleotide design The design of these oligonucleotides (ie, INVADER oligonucleotides and probes) is performed using standard methods in the art. For example, sequences that are self-complementary such that the resulting oligonucleotides fold themselves or hybridize to each other without binding to the target nucleic acid are usually not used.

One condition for choosing the length of these oligonucleotides is the complexity of the sample containing the target nucleic acid. For example, the human genome is approximately 3 × 10 ⁹ base pairs in length. A sequence of 10 nucleotides appears at a frequency of 1: 4 ^{10 in} an arbitrary base sequence, that is, a frequency of 1: 1,048,576, so there are about 2,861 times in 3 billion base pairs. Obviously, this length of oligonucleotide will have a low chance of specifically binding to a 10 nucleotide region in a target having the same length sequence as the human genome. However, if the target sequence was a 3 kb plasmid, there would be ample opportunities for such oligonucleotides to specifically bind. According to this same calculation method, a 16-base oligonucleotide (ie, a 16-mer) has a shortest sequence length and is likely to appear only once in 3 × 10 ⁹ base pairs. It is. This same degree of specificity means that the desired complex only occurs when two or more shorter oligonucleotides are in a cooperative form (ie, both or all bind to the target sequence of interest). Can be provided by such oligonucleotides, and the combination of short oligonucleotides provides the desired specificity. In one such embodiment, the cooperativity between short oligonucleotides is due to the coaxial stacking effect that can occur when the oligonucleotides hybridize to adjacent sites on the nucleic acid. In another embodiment, the short oligonucleotides are linked directly to each other or with one or more interstitial regions. Short oligonucleotides linked in this way can also be used to bind to regions away from the target or to bridge across regions of secondary structure within the target. Examples of such bridged oligonucleotides are described in WO 98/50403. This document is incorporated herein by reference in its entirety.

A second condition that must be considered in selecting the length of the oligonucleotide is the temperature range in which the oligonucleotide is considered to function. A 16-mer with an average base content (50% GC base) is calculated at approximately 41 ° C., depending on the concentration of the oligonucleotide and its target, the salt content of the reaction, and the exact sequence of nucleotides. _{Has m} value. In practice, longer oligonucleotides are selected to increase the specificity of hybridization. Oligonucleotides 20 to 25 nucleotides in length can be very specific if used in reactions performed at temperatures close to their calculated T _m s (range T _m to about 5 ° C) Often used because it is expensive. Furthermore, in the range of calculated T _m s from 50 to 70 ° C., such oligonucleotides (ie, 20-25 mer) are often catalyzed by thermostable enzymes that exhibit optimal activity near this temperature range. It can be used appropriately for the reaction.

  The maximum length of the selected oligonucleotide also depends on the desired specificity. It is too long to increase the risk of stable binding to partially complementary sequences, or cannot be easily released when desired (eg, cannot be dissociated from the target after cleavage occurs, or the enzyme in the reaction mixture) And lengths that cannot be dissociated at reaction temperatures suitable for other materials) should be avoided.

  The first step in designing and selecting oligonucleotides to perform INVADER oligonucleotide specific cleavage follows the general principles of these samples. Each oligonucleotide can be selected according to the guidelines listed above, each considered as a sequence specific probe. That is, each oligonucleotide generally has a length that can reasonably be expected to hybridize only to the target sequence of interest in a complex sample in the range of 20-40 nucleotides. Alternatively, since the INVADER oligonucleotide-specific cleavage assay relies on the co-operation of these oligonucleotides, the combined length of the two oligonucleotides that span / bind to the target is selected to be within this range Each individual oligonucleotide can range from about 13 to 17 nucleotides. If a non-thermal stable cutting means is used for the reaction, such a design can be used and the reaction must be carried out at a temperature lower than the temperature used when using the heat stable cutting means. In some embodiments, it is desirable to bind these oligonucleotides multiple times within a single target nucleic acid (eg, bind multiple variants within a target, or multiple similar sequences). The method according to the present invention is not limited to specific size probes or INVADER oligonucleotides.

  The second step in designing the oligonucleotide pair of this assay is to select how much the upstream “INVADER” oligonucleotide sequence overlaps with the downstream “probe” sequence, resulting in the size of the probe cleaved. This is a step of selecting whether to do. In some preferred embodiments, the probe oligonucleotide can be turned over. That is, the probe can be detached to bind and cleave another copy of the probe molecule without the need for denaturation by temperature or substitution by polymerization. In one embodiment of the present assay, exonucleolytic degradation by a cleaving factor can promote probe turnover, but in some preferred embodiments of the invention, this exonuclease activity is not required for turnover. . For example, in some embodiments, the reaction temperature and reaction conditions are selected to create an equilibrium state where the probe is hybridizing and dissociating to the target. In another embodiment, the temperature and reaction conditions for initiating binding of the unbound probe to the target strand and physically displacing the bound probe are selected. In yet another embodiment, the temperature and reaction conditions are selected such that either or both of the probe displacement mechanisms occur at any rate. The method according to the present invention is not limited to a specific probe replacement mechanism. When the probe binds to the target to form a cleavage structure, cleavage can occur by some mechanism. By continuously repeating the binding and detachment of the probe to the target, the binding and cleavage of the probe to each copy of the target nucleic acid can be performed multiple times.

i) Non-overlapping sequences The relationship between the 3 'end of the upstream oligonucleotide on the probe and the desired cleavage site must be carefully designed. A suitable site for cleaving the same type of structure-specific endonuclease used herein is known to be a single base pair in the duplex (Lyamichev et al., Supra). . Previously, it was thought that the presence of an upstream oligonucleotide or primer would allow the cleavage site to move from this preferred site to the single stranded region of the 5 ′ arm (Lyamichev et al., Supra, and US Pat. No. 5,442,253). issue). Compared to this previously proposed mechanism, without limiting the present invention to a particular mechanism, it is immediately 5 'or upstream of the cleavage site on a probe (such as a miniprobe and a medium length probe). It is believed that the nucleotide should be able to base pair with the target to produce efficient cleavage. In the case of the present invention, this is likely a nucleotide in the probe sequence immediately upstream of the intended cleavage site. Furthermore, as described above, in order to cleave at the same site in the probe, the upstream oligonucleotide must have a 3 ′ base (ie, nucleotide) immediately upstream of the target cleavage site of the probe. Don't be. In embodiments where the INVADER and probe oligonucleotide share a sequence overlap, this means that the 3 ′ terminal nucleotide of the upstream oligonucleotide and the 5 ′ side of the cleavage site of the probe oligonucleotide are linked to the nucleotide of the corresponding target sequence. You will be in competition for the pairing.

  In order to investigate the outcome of this competition (which base paired during successful cleavage), the probe and INVADER oligonucleotide were displaced so that the probe or INVADER oligonucleotide was I made a mismatch. The effect of both sequences on the cleavage rate was investigated. When the INVADER oligonucleotide did not base pair at the 3 ′ end, the cleavage rate was not reduced. However, removal of this base moved the cleavage site upstream of the site of interest. In contrast, when the probe oligonucleotide was not base paired with the target immediately upstream of the site where the INVADER oligonucleotide was commanding cleavage, the cleavage rate was significantly reduced. This suggests that the probe oligonucleotide is a base pairing molecule at this position.

  The 3 ′ end upstream of the INVADER oligonucleotide is not paired during the cleavage process, but is still considered important for accurate cleavage positioning. In order to find out what part of the 3 ′ terminal nucleotide is needed to determine the cleavage position, INVADER oligonucleotides with nucleotides modified in various ways, terminating at this position, were designed. The sugars examined include 2 ′ deoxyribose, dideoxyribose, 3 ′ deoxyribose, 2′O-methylribose, arabinose with a 3 ′ phosphate group, and arabinose with a 3 ′ phosphate group. Abasic ribose with 3 ′ phosphate and abasic ribose without 3 ′ phosphate were examined. Synthetic “universal” bases such as 3-nitropyrrole and 5-3 nitroindole on ribose sugars were investigated. Finally, acridine which is a base-like aromatic ring structure was examined. This examined what was previously a nucleotide without a sugar group but bound to the 3 'end. The results obtained confirm that the aromatic ring of the base (at the 3 'end of the INVADER oligonucleotide) is an important part for detecting cleavage at the desired site in the downstream probe. The 3 ′ end portion of the INVADER oligonucleotide need not be a base complementary to the target nucleic acid.

ii) Miniprobes and midiprobes As described above, INVADER oligonucleotide-specific cleavage assays were performed using INVADER and probe oligonucleotides having a length of approximately 13-25 nucleotides (typically 20-25 nucleotides). Can be used. It is also believed that the oligonucleotides themselves can be composed of short base sequences that align along the target strand but do not covalently bind. This means that there is a nick in the sugar-phosphate backbone of the composite oligonucleotide, but there is no failure in the base-paired nucleotide sequence in the resulting duplex. When a short strand of nucleic acid is aligned continuously along a longer strand, each hybridization is stabilized by the hybridization of adjacent fragments. This is because base pairing can be stacked along the helix as if the skeletons were actually continuous. This binding cooperativity provides stability to the interaction of each segment in the presence of an excess of what would be expected for a segment that hybridizes only to the longer oligonucleotide. One use for this observation is to use three hexameric oligonucleotides designed to hybridize in this way with primers for DNA sequencing, typically 18 nucleotides in length. (Kotler et al., Proc. Natl. Acad. Sci. USA, 90: 4241 (1993). The resulting double-nicked primer disrupts hexamer hybridization. However, 18-mer hybridization can be extended by an enzyme in a reaction performed at a temperature that is assumed not to decay.

  The conjugated or split oligonucleotide is suitably applied to INVADER-specific cleavage assay. For example, split a probe oligonucleotide into two oligonucleotides in a continuous and adjacent manner along the target oligonucleotide so that the downstream oligonucleotide (similar to a probe) can be assembled from two small fragments: Can do. That is, a short segment of 6 to 10 nucleotides (referred to as “miniprobe”) that is cleaved during the detection reaction, and an oligonucleotide that hybridizes immediately downstream of the miniprobe (referred to as “stacker”) And segments that help stabilize the hybridization of the probe. In order to form a cleavage structure, an upstream oligonucleotide (INVADER oligonucleotide) is provided to direct the cleavage activity to the desired region of the miniprobe. By assembling probes from unligated nucleic acid fragments (ie, miniprobes and stackers), regions of the sequence can be altered without requiring re-synthesis of the fully proven sequence. Therefore, the cost and flexibility of the detection system can be increased. In addition, when using unconjugated complex oligonucleotides, the system becomes more stringent for exact match hybridization to achieve signal generation and is sensitive to detect mutations or changes in the target nucleic acid sequence. It becomes possible to use as this method.

  In one embodiment, the method according to the present invention uses at least three types of oligonucleotides that interact with a target nucleic acid to form a cleavage structure for structure-specific nucleotides. Specifically, the cleavage structure is: i) a single-stranded or double-stranded target nucleic acid (when a double-stranded target nucleic acid is used, it can be made into a single strand by heating, for example); ii ) A first oligonucleotide, referred to as a “stacker”, which defines this region by being complementary to the first region of the target nucleic acid sequence; iii) a “miniprobe” A second oligonucleotide which defines this region by being complementary to the second region of the target nucleic acid sequence; iv) referred to as the “INVADER oligonucleotide” A third oligonucleotide whose 5 ′ portion defines a third region of the same nucleic acid sequence and whose second or 3 ′ portion overlaps a region defined by the second oligonucleotide. Third Includes oligonucleotides. As described above for embodiments that do not use a stacker, an overlapping region may mean a region where there is a physical overlap, rather than a sequence overlap, between the INVADER oligonucleotide and the probe oligonucleotide.

In addition to the advantages cited above, the use of a composite design for the oligonucleotides that form the cleavage structure allows greater freedom in designing reaction conditions for performing INVADER-specific cleavage assays. As noted above, when longer probes (eg, 16-25 nucleotides) are used for detection in reactions performed at temperatures below their T _m , the double strands that probe cleavage is part of It plays an important role in destabilizing and enables the turnover and reuse of recognition sites on target nucleic acids. In contrast, a reaction temperature above the _Tm of the probe means that the probe molecule hybridizes at a very fast rate and is released from the target even if the probe is not cleaved. When upstream INVADER oligonucleotides and cleavage factors are provided, the probe is specifically cleaved, but this cleavage is not necessary for probe replacement. When long probes (such as 16-25 nucleotides) are used in this way, the temperature required to achieve this state is high, approximately 65-70 ° C. for 25-mers with an average base composition It is. Since such a high temperature is required, the selection of cleaving factors is limited to those that have high thermal stability and can contribute to the background of the reaction due to thermal degradation of the probe oligonucleotide, depending on the detection method. Is done. With the miniprobe, this latter mechanism of probe replacement can be performed at low temperatures. Therefore, shorter probes are preferred for embodiments that use lower reaction temperatures.

  The miniprobe according to the present invention can be of various lengths depending on the desired application. In one embodiment, it may be short compared to standard probes (eg, 16-25 nucleotides) and may range from 6-10 nucleotides. When using such a short probe, reaction conditions can be selected such that mini-probe hybridization does not occur in the absence of a stacker oligonucleotide. In this way, short probes can have statistical specificity and selectivity of longer sequences. Loss of this cooperativity when disruptions occur in the cooperative binding of miniprobe and stacker nucleic acids, such as can occur due to mismatches in short sequences complementary to the target nucleic acid or in continuous double-stranded junctions. Thereby significantly reducing the stability of the short duplex (ie, the mini-probe duplex) and reducing the amount of cleavage product in the assay method of the present invention.

It is also contemplated that medium length probes can be used. Such probes are on the order of 11-15 nucleotides and can fuse with some of the features associated with long probes as described at the outset, such as being able to hybridize and cleave without the aid of stacker oligonucleotides. Can do. At temperatures below the expected T _{m of} such probes, the mechanism of turnover is similar to that studied for probes of around 20 nucleotides, removing sequences in overlapping regions for destabilization and rotation. Depends on.

Medium length probes can be used even at higher temperatures, and above the expected T _m , probe rotation can be facilitated by melting rather than cutting. However, in contrast to the longer probes described above, the temperature required to be able to take advantage of this temperature dependent turnover is lower (about 40-60 ° C), so Both the cutting means and the nucleic acid can be protected from thermal degradation. In this way, in some cases, medium length probes can act similarly to the miniprobes described above. Even more similar to a miniprobe is that depending on the reaction conditions, the presence of a stacker promotes the accumulation of cleavage signals from medium length probes.

  In short, standard length probes usually do not benefit from the presence of downstream stacker oligonucleotides (provided that such oligonucleotides also destroy target nucleic acid structures that inhibit probe binding). The probe can be used in conditions where several types of nucleotides need to be removed to efficiently release the oligonucleotide from the target. When manipulating probe exchange using reaction temperatures, it may be necessary to use temperatures with high risk of nucleic acid and enzyme pyrolysis for standard probes.

  Miniprobes are very short and work best when a stacker oligonucleotide is present downstream. Miniprobes are well suited to reaction conditions that use reaction temperatures that allow rapid probe exchange on the target, regardless of which base is cleaved. In a reaction with a sufficient amount of cleavage means, a probe that binds well should be cleaved quickly before it melts away.

  Medium length probes or miniprobes can be used in the same reaction as those suitable for long probes, combining the characteristics of these probes. In a preferred embodiment, the medium length probe is used at a sufficiently high temperature that the probe hybridizes to the target and releases rapidly with or without cleavage. Medium length probes can show higher efficiency in the presence of stackers under certain conditions.

  The distinction between miniprobes, midi (ie, medium length) probes, and long probes is constant and is not considered to be based on length alone. The efficiency of a given probe will vary depending on its specific sequence, choice of solution conditions, choice of temperature, and selected cutting means.

  The assembly of oligonucleotides comprising a cleavage structure according to the present invention senses mismatches between the probe and the target. It is also believed that the target sequence involved can be distinguished using the mismatch between the INVADER oligonucleotide and the target. In 3-oligonucleotide systems, including INVADER, probes, and stacker oligonucleotides, mismatches may be present in any region of the duplex formed between these oligonucleotides and the target It is done. In preferred embodiments, the detected mismatch is present in the probe. In a particularly preferred embodiment, the mismatch is present in a base pair in the probe that is immediately upstream (ie, 5 ') to the site that is cleaved when the probe does not match the target.

  In another preferred embodiment, the mismatch to be detected is in a region defined by miniprobe hybridization. In a particularly preferred embodiment, the mismatch is present in the miniprobe at a base pair that is immediately upstream (ie, 5 ') to the site that is cleaved when the miniprobe does not match the target.

iii) Software for designing oligonucleotides for INVADER assays The present invention provides systems and methods for designing oligonucleotides for use in detection assays. In particular, the present invention successfully hybridizes to an appropriate region of a target nucleic acid (eg, a target nucleic acid region that does not include secondary structure) under reaction conditions desirable for a detection assay (eg, temperature, buffer conditions, etc.). Systems and methods for designing oligonucleotides for soy are provided. These systems and methods allow a number of different oligonucleotides (eg, hybridize to different sites on a target nucleic acid) that all function in the same or under the same reaction conditions. It is also possible to design oligonucleotides that hybridize to two or more different target nucleic acids. These systems and methods can also be used to design control samples that function under experimental conditions.

  The systems and methods according to the present invention are not limited to a particular detection assay, and the following description includes an INVADER assay (Third Wave Technologies, Madison, Wis.) For detecting SNPs. (Madison, WI); see, for example, US Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, and WO 97/27214 and WO 98/42873. , Which are incorporated herein by reference), which specifically describe the invention. Those skilled in the art will recognize the specific and general features of the present illustrative example as to other detection assays and INVADER assays with a purpose other than SNP detection (eg, DNA or RNA quantification, RNA splice sites). It can be understood that it can be widely applied to use when designing detection etc.). In addition, all of the algorithms described herein can be applied as separate software components, or to design INVADER assay probe sets without using the INVADERCREATIOR design system described below. It can also be calculated manually.

Design of oligonucleotides for INVADER assay using INVADERCREATIOR program
In some embodiments of designing an oligonucleotide for use in an INVADER assay to detect SNPs, the sequence of interest can be transferred to the INVADERCREATIOR program (Third Wave Technologies, Madison, Wis.). )). As mentioned above, for analysis from several sources, it can be entered directly into the computer hosting the INVADERCREATIOR program or arranged via a remote computer connected by a communication network (eg a LAN, Intranet, or Internet network) Enter. With this probe, a sense strand and an antisense strand can be designed. The chains are generally selected based on ease of synthesis, minimization of secondary structure formation, and ease of manufacture. In some embodiments, the chain of sequences to be designed by the user is selected. In another embodiment, the software automatically selects a strand. By incorporating the thermodynamic parameters (Allawi and SantaLucia, Biochemistry, 36: 10581 [1997]) for optimal probe rotation and signal generation, oligonucleotide probes can be designed to select preselected measurement temperatures (eg, , 63 ° C). Based on these criteria, a final set of probes (eg, primary probes for two alleles and one INVADER oligonucleotide) are selected.

  In some embodiments, the INVADERCREATIOR system can be used on the BLAST (National Center for Biotechnology Information, National Library of Medicine, National Institute of Health) website. ) RNAstructure (Mathews et al., RNA 5: 1458 [), a software program that incorporates reliable site access, including links to, and mfold (Zuker, Science, 244: 48 [1989]) 1999]) is a web-based program with reliable site access that can be linked to. RNAstructure tests oligonucleotide design proposals created by INVADERCREATIOR for the possibility of single molecule and bimolecular complex formation. INVADERCREATIOR supports Open Database Connectivity (ODBC) and uses an Oracle (Oracle) database for export / integration. Most Genome Centers use UNIX, so the INVADERCREATIOR system has an Oracle built-in to work well with UNIX systems.

  In some aspects, INVADERCREATIOR analysis is provided on a separate server (eg, a Sun server) so that it can handle the analysis of large batch jobs. For example, a customer can submit up to 2,000 SNP sequences in one email. The server passes the batch of sequences to the INVADERCREATIOR software and, when the program is started, designs a set of SNPs. In some embodiments, the probe set design is returned to the user within 24 hours of receiving the sequence.

  In some preferred embodiments, each INVADER assay reaction includes two or more target-specific, unlabeled oligonucleotides, upstream INVADER oligonucleotides and downstream probe oligonucleotides for the primary reaction. INVADER oligonucleotides are generally designed to bind stably at reaction temperatures, probes are designed to freely associate and dissociate with the target strand, and overlap with uncut probes It is designed so that cleavage occurs only when hybridized adjacent to the INVADER oligonucleotide. In some embodiments, the probe is a 5 ′ flap or “arm” that is not complementary to the target, and the flap is released from the probe when cleavage occurs. In some embodiments, the released flap participates in the secondary reaction as an INVADER oligonucleotide.

  The following discussion provides an example of how a user interface for the INVADERCREATIOR program can be configured.

  The user opens a work screen (eg, by clicking an icon on a computer desktop screen (eg, the Windows desktop)) (FIG. 42), and information related to the target sequence for which the assay is to be designed. Enter. In some embodiments, the user enters a target sequence. In another embodiment, the user enters a code or number so that sequences can be captured from the database. In yet another aspect, additional information can be provided, such as the user's name, a recognition number attached to the target sequence, and / or a receipt number. In a preferred embodiment, the user indicates whether the target nucleic acid is DNA or RNA (eg, via a checkbox or drop-down menu). In another preferred embodiment, the user indicates the species from which the nucleic acid is derived. In particularly preferred embodiments, the user detects whether the design is a single sequence (ie, one target sequence or allele per reaction) or multiple sequences (ie, multiple target sequences or alleles per reaction) Tell what to do. Once the necessary selections and inputs have been made, the user starts the analysis process. In one embodiment, the user presses the button “Go Design It” to continue the process.

  In some embodiments, the software authenticates the input to the field before performing the process. In some embodiments, the software verifies that all types of information are fully entered in all fields. In another embodiment, the software verifies that the input sequence meets the selected requirements (eg, minimum or maximum length, DNA or RNA content). If any field is not entered correctly, an error message or dialog box appears. In a preferred embodiment, the error message indicates which fields are incomplete and / or inaccurate. Once the sequence entry is confirmed, the software begins assay design.

  In some aspects, the information entered in the order entry field identifies what type of design should be created. In a preferred embodiment, the target sequence and multiple sequence checkboxes specify what type of design should be created. Design options include SNP assays, multiplex SNP assays (eg, when combining probe sets for different alleles in a single reaction), multiple SNP assays (eg, multiple ANP assays: eg, input) Such as, but not limited to, multiple probe arm assays) and the like.

  In some aspects, the INVADERCREATIOR software is initiated by a Web Order (WebOE) process (ie, by an intra / Internet browser interface) and these parameters are not entered by a menu or checkbox, but by WebOE From the applet via the <param> tag.

  For multiple SNP designs, the user selects two or more designs to use. In some embodiments, selecting this opens a new screen (eg, multiple SNP design selection screen, FIG. 43). In some embodiments, the software creates a design for each locus in the target sequence, gives each score, and displays them on this screen. The user can then select two designs to use. In some embodiments, the user selects the first and second designs (eg, via a menu or button) and clicks the button “Go Design It” to continue working.

To select probe sequences that work optimally at a given reaction temperature, the nearest-neighbor model and published DNA duplex formation parameters (Allawi and SantaLucia, Biochemistry, 36: 10581 [1997]) Is used to calculate the melting temperature (T _m ) of the SNP to be detected. In embodiments where the target strand is RNA, parameters suitable for RNA / DNA heteroduplex formation can be used. Calculated because the salt concentration for the assay is often different from the solution conditions (1 M NaCl and no divalent metal) from which adjacent parameters were obtained, and the presence and concentration of the enzyme affects the optimal reaction temperature The measured T _m value must be adjusted to determine the optimum temperature at which the reaction should be carried out. One way to compensate for these factors is to change the value provided for the salt concentration when calculating the melting temperature. This adjustment is called “salt correction”. As used herein, the term “salt correction” refers to the salt concentration in order to reflect the effect of parameters or conditions other than salt that affect the nucleic acid duplex on the calculation of the T _m value for the nucleic acid strand. It means changing the provided value. Changing the value provided for the concentration of the nucleic acid strand also affects these calculation results. By using a value of 0.5 M NaCl (SantaLucia, Proc. Natl. Acad. Sci. USA, 95: 1460 (1998)) and a nucleic acid chain concentration of about 1 mM of the probe and 1 fM of the target, the probe-target melting temperature The algorithm used to calculate can be used to predict the optimal reaction temperature for the INVADER assay. For the 30 probe sets, the average deviation between the optimal assay temperature calculated by this method and the optimal assay temperature determined by experiment is about 1.5 ° C.

The length of the analyte-specific region (ASR) of the downstream probe is defined by the temperature selected to perform the reaction (eg, 63 ° C.). Starting from the position of the mutated nucleotide on the target DNA (target base pairing to probe nucleotide 5 'of the target cleavage site), adding to the 3' end, the calculated optimal reaction temperature that matches the desired reaction temperature ( An iterative method is used in which the length of the target binding region of the probe is increased by one base pair at a time until T _m + a salt correction value that compensates for the action of the enzyme. Preferably, non-complementary arms of the probe are selected to allow the secondary reaction to be repeated at the same reaction temperature. Dimeric complexes that can interfere with the reaction using probes such as mfold (Zuker, Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads, Nucleic Acids Res., 17: 8543 [1989]) All probe oligonucleotides are screened for the possibility of secondary structure formation. The design of INVADER oligonucleotides follows the same principle. Briefly, starting from the N position on the target DNA, the 3 ′ end of the INVADER oligonucleotide is designed to have nucleotides that are not complementary to either allele that would be included in the test sample. Mismatches do not adversely affect cleavage (Lyamichev et al., Nature Biotechnology, 17: 292 [1999]) and can facilitate probe rotation, possibly by minimizing coaxial stabilization effects. Additional residues complementary to the target DNA, starting at position N-1, until the stability of the hybrid of the INVADER oligonucleotide and the target exceeds the stability of the probe (and consequently the planned assay reaction) Over temperature), generally add in the 5 'direction until it exceeds 15-20 ° C.

  In some embodiments, the cleaved fragments released from the primary reactant are used in the secondary reaction. One feature of the assay design is that all of the probe sequences can be selected so that the primary and secondary reactions occur at the same optimal temperature so that the reaction steps can be performed simultaneously. In an alternative embodiment, the probe can be designed to operate at different optimum temperatures so that the reaction steps do not occur simultaneously at the optimum temperature.

  In some aspects, the software provides the user with an opportunity to change various design aspects, including but not limited to: That is, optimal temperatures and concentrations of probes, targets and INVADER oligonucleotides, protecting groups, probe arms, dyes, capping groups and other additional groups, probe and target bases (e.g., missing bases from targets and / or probes) Lost or added, or change the internal base of INVADER and / or probe and / or target oligonucleotide). In some embodiments, changes can be made by selecting from a menu. In a preferred embodiment, this selection opens a new screen (eg, designer worksheet screen, FIG. 44).

  In some embodiments, the software provides a scoring system for displaying assay design quality (eg, likelihood of efficiency). In one embodiment, the scoring system includes an initial score score that represents an ideal design (eg, 100 points) and is known or suspected to have a detrimental effect on assay efficiency. This is a system in which penalty values are assigned to design features. Penalty values can vary depending on the type of assay being designed (e.g., single-stranded, multi-stranded), and the temperature at which the assay reaction is performed, but not limited to these parameters in the assay other than the sequence. Can be changed. The following examples provide exemplary scoring criteria for use with some aspects of the INVADER assay based on information specified by experiments. Examples of design features that can be penalized for scoring include, but are not limited to: [Penalty values are shown in parentheses. The first number is for a low temperature assay (eg, 62-64 ° C.) and the second number is for a high temperature assay (eg, 65-66 ° C.).

2．[70:70] プローブが多型性を含む5塩基の直列配列（すなわち、一列になった5個の同じ塩基）を有する
3．[60:60] プローブが多型性に隣接した5塩基の直列配列を有する
4．[50:50] プローブが多型性から1塩基のところに5塩基の直列配列を有する
5．[40:40] プローブが多型性から2塩基のところに5塩基の直列配列を有する
6．[50:50] プローブの5塩基の直列配列がGである−追加的ペナルティー
7．[100:100] プローブがいずれかに6塩基の直列配列を有する
8．[90:90] 2塩基または3塩基の配列が4回以上繰り返される
9．[100:100] 縮重塩基がプローブ中に存在する
10．[60:90] プローブがハイブリダイズする領域が短い（65〜67℃用の設計では13塩基以下；62〜64℃用の設計では12塩基以下）
11．[40:90] プローブがハイブリダイズする領域が長い（65〜67℃用の設計では29塩基以下；62〜64℃用の設計では28塩基以下）
12．[5:5] プローブがハイブリダイズする領域の長さ−一塩基付加される毎のペナルティー
13．[80:80] プローブのアームの後ろに来る最初の3塩基を区別しにくいIns/Del設計
14．[100:100] プローブ標的の7.5℃の範囲内にあるINVADERオリゴヌクレオチドの計算上のT_m値（65〜67℃用の設計ではINVADERオリゴヌクレオチドが70.5℃以下；62〜64℃用の設計ではINVADERオリゴヌクレオチドが69.5℃以下）
15．[20:20] プローブの計算上のT_m値が2.0℃以上差がある
16．[100:100] プローブが、標的のT_m値と比べて2℃より低い計算上のT_m値をもつ
17．[10:10] 標的の一方の核酸鎖が、もう一方の鎖よりも8塩基長い
18．[30:30] INVADERオリゴヌクレオチドがいずれかに6塩基の直列配列を有する−初期ペナルティー
19．[70:70] INVADERオリゴヌクレオチドの6塩基の直列配列がGである−追加的ペナルティー
20．[15:15] プローブがハイブリダイズする領域が、14、15もしくは24〜28塩基長（65〜67℃）、または13、14もしくは26、27塩基長い（62〜64℃）
21．[15:15] プローブが、多型を含むGの4塩基の直列配列を有する 1． [100: 100] The 3 ′ end of the INVADER oligonucleotide is similar to the probe arm:

2． [70:70] Probe has a 5 base tandem sequence (ie, 5 identical bases in a row) containing polymorphisms
3． [60:60] Probe has a 5 base tandem sequence adjacent to the polymorphism
Four. [50:50] The probe has a 5 base tandem sequence at 1 base due to polymorphism.
Five. [40:40] Probe has a 5 base tandem sequence at 2 bases due to polymorphism
6． [50:50] The 5 base tandem sequence of the probe is G-additional penalty
7． [100: 100] Probe has a 6 base tandem sequence in either
8． [90:90] Sequence of 2 or 3 bases is repeated more than 4 times
9． [100: 100] Degenerate base is present in the probe
Ten. [60:90] Probe hybridization region is short (13 bases or less for the 65-67 ° C design; 12 bases or less for the 62-64 ° C design)
11． [40:90] Long probe hybridization region (29 bases or less for 65-67 ° C design; 28 bases or less for 62-64 ° C design)
12． [5: 5] Length of probe hybridizing area-penalty for every single base addition
13. [80:80] Ins / Del design makes it difficult to distinguish the first three bases behind the probe arm
14. [100: 100] Calculated T _m values for INVADER oligonucleotides within 7.5 ° C of the probe target (INVADER oligonucleotides are below 70.5 ° C for 65-67 ° C designs; for 62-64 ° C designs INVADER oligonucleotide is 69.5 ° C or less)
15． [20:20] Probe _Tm value is more than 2.0 ℃ difference
16． [100: 100] The probe has a calculated T _m value lower than 2 ° C compared to the target T _m value
17． [10:10] One nucleic acid strand of the target is 8 bases longer than the other strand
18． [30:30] INVADER oligonucleotide has a 6-base tandem sequence in either-initial penalty
19. [70:70] 6-base tandem sequence of INVADER oligonucleotide is G-additional penalty
20． [15:15] Probe hybridization region is 14, 15, or 24-28 bases long (65-67 ° C), or 13, 14 or 26, 27 bases long (62-64 ° C)
twenty one. [15:15] Probe has a 4 base tandem sequence of G including polymorphism

  In a particularly preferred embodiment, the temperature for each oligonucleotide in the design is recalculated by computer and the score is recalculated by computer each time it is changed. In some embodiments, clicking on the “Description” button will allow you to see an explanation of the scoring. In some embodiments, BLAST search options are provided. In a preferred embodiment, the BLAST search is performed when the “BLAST design” button is clicked. In some embodiments, this operation displays a dialog box describing the BLAST process. In a preferred embodiment, the results of the BLAST search are displayed in a highlighted form on the designer worksheet.

  In some embodiments, the user clicks the “accept” button to accept the design. In another aspect, the design is accepted without user intervention. In a preferred embodiment, the program sends the approved design to the next processing stage (eg, creation, file or database). In some embodiments, the program is illustrated on a screen (eg, output page, FIG. 45), allowing the final design created to be reviewed and the design to be annotated. In a preferred embodiment, the user can return to the designer worksheet (eg, by clicking on a “back” button). Alternatively, the design can be saved (eg, by clicking a “Save” button) and continued (eg, to present the designed oligonucleotide for production).

  In some embodiments, the program creates a screen that displays a blueprint that is optimized for printing (eg, display of text only) or for other output (eg, output screen, FIG. 46). Present options. In a preferred embodiment, the output screen provides a description of a design that is particularly suitable for printing or outputting to another application (eg, copying and pasting to another application). In a particularly preferred embodiment, the output screen opens in a separate window.

  The present invention is not limited to the use of INVADERCREATIOR software. In fact, there are GCG Wisconsin packages (Genetics computer Group, Madison, Wis.), And Vector NTI (Informax, Rockville, Maryland). Various software programs have been devised and marketed.

b) Design of reaction conditions Target nucleic acids (eg RNA and DNA) can be analyzed using the method according to the present invention using a 5 'nuclease or other suitable cleavage factor. Such nucleic acids can be obtained using standard molecular biology techniques. For example, nucleic acids (RNA or DNA) can be isolated from tissue samples (eg, biopsy specimens), tissue culture cells, samples containing bacteria and / or viruses (such as bacterial and / or viral cultures), and the like. it can. The target nucleic acid can also be transcribed in vitro from a DNA template, chemically synthesized, or amplified by the polymerase chain reaction. In addition, nucleic acids can be isolated from organisms as genomic material or as plasmids or similar extrachromosomal DNA, or such enzymes produced by treatment with restriction enzymes or other cleavage factors, or shear forces. It may be a fragment of the substance. Fragments can also be synthesized.

  When the target, probe, and INVADER oligonucleotide nucleic acid are collected and added to the cleavage reaction according to the present invention, dideoxynucleotide sequencing and polymerase chain reaction (PCR), which are widely used in the design of enzyme assays using oligonucleotides ) And other principles are used. When performed in these assays, a sufficient excess of oligonucleotide is provided so that the rate of hybridization to the target nucleic acid is very high. These assays are usually performed with 50 to 2 picomoles of each oligonucleotide added per microliter of reaction mixture. However, it is not limited to this range. In the examples described herein, amounts of oligonucleotide from 250 femtomole to 5 pmoles per microliter of reaction volume were used. These values were chosen for ease of demonstration and are not intended to limit the practice of the invention to these concentrations. Other (eg, low) oligonucleotide concentrations used in other molecular biological reactions are also contemplated.

The INVADER oligonucleotide is preferably one that can be readily used to cleave each probe oligonucleotide that hybridizes to the target nucleic acid. In some of the embodiments described herein, the INVADER oligonucleotide is provided in excess of the probe oligonucleotide. This is an effective way to make the INVADER oligonucleotide readily available in such embodiments, but the practice of the invention is limited to the condition that the INVADER oligonucleotide is in excess of the probe. Nor is it limited to INVADER: probe at a certain ratio (eg, in some of the embodiments described herein, the probe is in excess of the INVADER oligonucleotide). Provided). Another way to ensure that the INVADER oligonucleotide is present whenever the probe binds to the target nucleic acid is to design the INVADER oligonucleotide to more stably hybridize to the target, i.e. than the probe. Is to have a high T _m value. This can be done by any of the methods for increasing nucleic acid duplex stability discussed herein (eg, increasing the degree of complementarity to the target nucleic acid).

Buffer conditions that are compatible with both oligonucleotide / target hybridization and cleavage factor activity should be selected. Optimal buffer conditions for nucleic acid modifying enzymes, particularly DNA modifying enzymes, generally include sufficient amounts of monovalent and divalent salts to associate nucleic acid strands by base pairing. When the method according to the invention is carried out with an enzymatic cleavage factor not specifically described herein, the reaction is generally reported to be optimal for the nuclease function of the cleavage factor. Can be done with liquid. In general, to determine whether a cleaving factor is available in the method, the cleaving factor in question is either MOPS / MnCl ₂ / KCl buffer, or the Mg-containing buffer described herein, And the test reaction is carried out in any buffer reported in the manufacturer's data sheet, article or personal report as suitable for use of the factor.

  The product of the INVADER oligonucleotide-specific cleavage reaction is a fragment generated by structure-specific cleavage of the input oligonucleotide. The resulting cleaved and / or uncleaved oligonucleotides can be electrophoresed, chromatographed, fluorescence polarized, mass spectrometry, and on-chip (on various supports such as acrylamide or agarose gels, paper, etc.) There are methods such as hybridization, but can be analyzed and analyzed by a number of methods including but not limited to these. In some embodiments, the present invention is specifically described using electrophoretic separation to analyze cleavage reaction products. However, the analysis method of the cleavage product is not limited to electrophoresis. Electrophoresis is widely practiced in the art and is readily available to the average practitioner, so electrophoretic methods are used to illustrate the method according to the present invention. Selected. As another example, the invention is shown without electrophoresis or any other degradation of the cleavage product.

Probes and INVADER oligonucleotides can contain labels that aid in their detection after the cleavage reaction. The label is a radioisotope placed at the 5 ′ or 3 ′ end of the oligonucleotide (eg, a nucleotide labeled with ³² P or ³⁵ S), or a label distributed throughout the oligonucleotide (ie, A uniformly labeled oligonucleotide). The label may be a detectable moiety other than an isotope, such as a fluorophore that can be detected directly or a reactive group that can be specifically recognized by a secondary agent. For example, biotinylated oligonucleotides can be detected by searching with a streptavidin molecule conjugated to an indicator reagent (eg, alkaline phosphatase, or fluorophore) or using a specific antibody conjugated to a similar indicator. Thus, haptens such as dioxygenin can be detected. The reactive group can also be a specific configuration or sequence of nucleotides, such as another nucleic acid that can bind to or otherwise react with a secondary factor, and an enzyme, antibody, or the like. In some embodiments, the probe can be labeled with a fluorescent component and a quenching component, in which case cleavage of the cleavage structure separates the fluorescent component from the quenching component, resulting in a detectable signal (eg, FRET Detection method). In some embodiments, a change in the quenching of a signal from the donor fluorophor is detected, while in another embodiment, a change in luminescence from the acceptor fluorophore is detected. In yet another embodiment, the effect of FRET on both donor and acceptor luminescence is detected.

In some embodiments of the FRET detection method, the fluorescence lifetime of the fluorophore is measured (eg, time-resolved fluorescence). Although embodiments of time-resolved fluorescence detection methods are not specifically limited to labeling systems, examples of useful tags for time-resolved FRET measurements include europium chelates (Eu ³⁺ ; Biosclair et al., J. Biomelecular Screening 5 (5 ): 319 [2000]), europium trisbipyridine cryptate (TBPEu ³⁺ ; Alpha-Bazin et al., Anal. Biochem. 286 (1): 17 [2000]), and a ruthenium ligand complex ([ Ru (bpy) 2 (phen-ITC)] ²⁺ ; Youn et al., Anal. Biochem. 231 (1): 24 [1995]; Lakowicz et al., Anal. Biochem. 288: 62 [2001]).

c) Optimization of reaction conditions
INVADER oligonucleotide specific cleavage reactions are useful for detecting the presence of specific nucleic acids. In addition to the conditions described above for selection and design of INVADER oligonucleotides and probe nucleotides, the conditions under which the reaction is performed can be optimized to detect specific target sequences.

  One goal of optimizing the INVADER oligonucleotide specific cleavage assay is to enable specific detection even when the target nucleic acid copy number is the lowest. To achieve this goal, it is desirable to combine the reaction components to interact with maximum efficiency so that the reaction rate (eg, cleavage per minute) is maximized. Components that contribute to the overall efficiency of the reaction include hybridization rate, cleavage rate, and release efficiency of the cleaved probe.

  The rate of cleavage is a function of the selected means of cleavage and is optimized according to the manufacturer's instructions when using commercially available enzyme preparations or as described in the examples herein. be able to. Since other factors (hybridization rate, release efficiency) depend on how the reaction is carried out, optimization of these factors is described below.

  The three components of the cleavage reaction that significantly affect the nucleic acid hybridization rate are the nucleic acid concentration, the rate at which the cleavage reaction is performed, and the salt concentration and / or other charge-shielding ion concentration in the reaction solution.

  The concentration of oligonucleotide used in this type of assay is well known in the art and is as described above. An example of a common way to optimize oligonucleotide concentration is to select the starting amount of oligonucleotide for pilot testing, and many methods that use nucleotides use a concentration range of 0.01 to 2 μM . If an initial cleavage reaction has been performed, is the data being run in the absence of the target nucleic acid substantially free from the cleavage product; the cleavage site is a specific location according to the design of the INVADER oligonucleotide Ask whether the specific cleavage product can be easily detected in the presence of an uncleaved probe (or whether the amount of uncleaved substance overwhelms the chosen visualization method) be able to.

  A negative answer to these questions suggests that the probe concentration is too high and a series of reactions must be performed with serial dilutions of the probe until the appropriate amount is confirmed. Is done. If an appropriate amount for a given target nucleic acid is confirmed in a given sample type (eg, purified genomic DNA, body fluid extract, disrupted bacterial extract), re-optimization is not necessary. The type of sample is important because the complexity of the material present can affect the optimal conditions for probe concentration.

  Conversely, if the concentration of the starting probe selected is too low, the efficiency of hybridization will deteriorate and the reaction will be slow. When testing with increasing amounts of probe, concentration points where the concentration exceeds the optimal amount (eg, concentration points that produce undesirable effects such as background cleavage independent of the target sequence, or inhibition of cleavage product detection). Can be confirmed. Although hybridization is facilitated by excess probe, it is desirable, but not necessarily, to perform the reaction using a probe concentration that is slightly below this point.

  The concentration of INVADER oligonucleotide can be selected based on the conditions discussed for the above design. In some embodiments, the INVADER oligonucleotide is present in excess over the probe oligonucleotide. In a preferred embodiment, the probe oligonucleotide is present in excess over the INVADER oligonucleotide.

  Temperature is also an important factor in oligonucleotide hybridization. The temperature range studied depends, in part, on the design of the oligonucleotide, as described above. When it is desired to perform the reaction at a specific temperature (for example, due to the necessity of an enzyme, for convenience, for compatibility with a measurement device or a detection device), the oligonucleotide functioning in the reaction is optimally operated at the desired reaction temperature. Can be designed to Each INVADER reaction includes two or more sequence-specific oligonucleotides for the primary reaction: an upstream INVADER oligonucleotide and a downstream probe oligonucleotide. In some embodiments, the INVADER oligonucleotide is designed to bind stably at the reaction temperature, but the probe is designed to freely bind to and dissociate from the target and is non- Cleavage occurs only when the cleavage probe hybridizes adjacent to the overlapping INVADER oligonucleotide. In a preferred embodiment, a 5 'flap that is not complementary to the target is included so that when cleavage occurs, this flap is released from the probe. The released flap can be detected directly or indirectly. In some embodiments, the released flap participates in the secondary reaction as an INVADER oligonucleotide, as detailed below.

  Conditions that optimize the INVADER assay are generally those that allow specific detection of a minimal amount of target nucleic acid. Such conditions include the conditions that give the strongest target-dependent signal within a given time frame, or for a given amount of nucleic acid, or the fastest probe cleavage rate (ie, a probe that is cleaved per minute). It has the feature of enabling conditions.

As mentioned above, the cleavage factor concentration can affect the temperature that is actually optimal for the cleavage reaction. In addition, different cleaving factors can have different effects on the optimal reaction temperature, even when used at the same concentration (eg, the calculated T _m value and the observed optimal reaction temperature). The difference may be greater for some enzymes than others. Determining the appropriate salt correction for reactions using different enzymes or enzyme concentrations, or for other changes made to the reaction conditions, involves the following two steps. a) the step of measuring the optimal reaction conditions under the new reaction conditions and the salt concentration within the range of the T _m value algorithm for calculating the calculated temperature value that is in agreement with or very close to the observed optimal conditions The process of changing. Measurement of the optimal reaction temperature generally allows an increase in efficiency to be observed as it approaches the optimal temperature (by increasing or decreasing the temperature), and a decrease in efficiency is observed after the optimal temperature is exceeded. This includes performing the reaction at a temperature range selected so that the optimal temperature and temperature range can be identified (Lyamichev et al., Biochemistry, 39: 9253 [2000]).

  In some embodiments, a secondary reaction is utilized when the cleaved fragments released from the primary reaction hybridize to the synthesis cassette to form a secondary cleavage reaction. In some embodiments, the cassette includes a fluorescent component and a quenching component, and cleavage of the secondary cleavage structure separates the fluorescent component from the quenching component, resulting in a detectable signal (eg, a FRET detection method). Secondary reactions can be designed in various ways. For example, in some embodiments, the synthesis cassette comprises two types of oligonucleotides: an oligonucleotide containing a FRET component, and an INVADER oligonucleotide (ie, a flap released from a primary reactant) and a FRET oligonucleotide. Includes FRET / INVADER oligonucleotide crosslinking oligonucleotides that are hybridized to form a cleavage structure. In some embodiments, the synthesis cassette is provided as a hairpin structure (ie, a FRET oligonucleotide is linked by a loop to a bridging oligonucleotide at its 3 ′ end). This loop may be a nucleic acid or a spacer or linker other than a nucleic acid. The bound molecules can be collectively referred to as a FRET cassette. In the secondary reaction using the FRET cassette, the flap released from the secondary reaction acts as an INVADER oligonucleotide, but needs to be able to freely bind to and dissociate from the FRET cassette. By doing so, the released flaps can cut multiple FRET cassettes. Selecting all probe sequences so that these reactions occur at the same optimal temperature so that the primary and secondary reaction steps are performed simultaneously is an aspect of the assay design. In an alternative embodiment, the probe can be designed to operate at different optimum temperatures so that the reaction steps are not performed simultaneously at the optimum temperature. As described above, the same iterative process used to select the ARS of the probe can be used when designing the portion of the primary probe that participates in the secondary reaction.

  Another factor that determines the efficiency of hybridization is the salt concentration of the reaction. In many cases, the choice of solution conditions will depend on the conditions of the cleaving factor and the conditions of the purchased commercial reagents, so the manufacturer's instructions are the source of such information. When developing an assay that utilizes a particular cleavage factor, it is necessary to optimize the oligonucleotides and temperature described above under the buffer conditions most suitable for that cleavage factor.

  In some embodiments, additional factors can be included in the reaction mixture to increase the efficiency of the assay. For example, charged compounds such as aminoglycosides and other polyamines have been used to alter the conformation and function of DNA and RNA (eg Earnshaw and Gait, Nucl. Acids. Res. 26: 5551 [1998]; Robinson And Wang, Nucl. Acids. Res. 24: 676 [1996]; Jerinei, J. Mol. Biol. 304 (5): 707 [2000]; Schroeder et al., EMBO 19 (1): 1 [2000]). By including the aminoglycoside antibiotic neomycin sulfate (eg, 1 μM in the primary reaction), for example, by reducing the background signal and reducing the detection limit of a particular INVADER assay probe set, etc. Assay efficiency can be increased. Compounds of this type that can be utilized in INVADER assay reactions include, but are not limited to, aminoglycosides, olicomomerized aminoglycosides, and aminoglycoside bioconjugates, and spermine and hexaamine cobalt. Of polyanions.

  The “no enzyme” control can assess the stability of labeled oligonucleotides under specific reaction conditions or in the presence of the sample to be tested (eg, assessing samples for contaminating nucleases). It becomes possible. In this way, the substrate and the oligonucleotide are placed in a test tube containing all reaction components other than the enzyme and treated in the same manner as the reaction containing the enzyme. Another control can be included. For example, reactions with all components other than the target nucleic acid are useful to confirm the dependence of cleavage on the presence of the target sequence.

d) Selection of cleavage factors As shown in some examples, some 5 'nucleases do not require upstream oligonucleotides that are active in the cleavage reaction. Cleavage may be slow without the upstream oligonucleotide, but cleavage still occurs (Lyamichev et al., Science 260, 778 [1993]; Kaiser et al., J. Biol. Chem., 274: 21387 [1999]). When the DNA strand is the template or target sequence to which the probe oligonucleotide hybridizes, several flap endonucleases (FEN) overlap, such as 5 'nuclease from DNA polymerase and FEN from Methanococcus yanasi Full cleavage can be performed without upstream oligonucleotides providing sites (Lyamichev et al., Science 260, 778 [1993]; Kaiser et al., J. Biol. Chem., 274: 21387 [1999], and US Pat. 5,843,669). Which are incorporated herein by reference in their entirety). These nucleases are some aspects of the INVADER assay, such as where cleavage of the probe in the absence of INVADER oligonucleotide yields another cleavage product that does not interfere with the intended analysis, or Selected for use in embodiments that allow both types of cleavage to occur, such as INVADER oligonucleotide-specific cleavage and INVADER oligonucleotide-independent cleavage.

  In another embodiment, an enzyme with this requirement will be used because it is preferred that the cleavage of the probe is dependent on the presence of an INVADER oligonucleotide upstream. Other FENs, such as FEN from Archaeoglobus fulgidus (Afu) and Pyrococcus furiosus (pfu), do not duplicate structures on DNA targets (ie, upstream structures). It can be considered to have an absolute requirement for duplication in nature (Lyamichev et al., Nat) because it cleaves at a faster rate than cleaving (with no oligonucleotide or with non-overlapping upstream oligonucleotide). Biotechnol., 17: 292 [1999]; Kaiser et al., J. Biol. Chem., 274: 21387 [1999]). When an RNA target hybridizes to a DNA oligonucleotide probe to form a cleavage structure, many FENs cleave downstream DNA probes only slightly, with or without duplication. In structures containing such RNA, the 5 ′ nuclease from DNA polymerase has a strong requirement for duplication and is essentially inactive in the absence of it. The selection of the enzyme used to detect the RNA target is discussed in detail in Section IV: Improved Enzymes Used in INVADER Oligonucleotide Specific Cleavage Reactions Containing RNA Targets below.

e) Search for multiple alleles
The INVADER oligonucleotide specific cleavage reaction is also useful for detecting and quantifying each variant or allele in a mixed sample population. As an example, such a need exists when analyzing cancer samples for mutations in genes associated with cancer. Biopsy samples from cancer may contain material that significantly supplements normal cells, so even if less than 5% of the target nucleic acid is present in a sample, mutations It is desirable to detect. In this case, it is also desirable to determine which fraction of the population has the mutation. There is no intention to limit the method according to the present invention to the analysis of cancer because a similar analysis can be performed to examine mutations in other gene systems.

  As shown below, in one embodiment, a mismatch due to a single base difference will inhibit probe cleavage, but will react under conditions where similar probes that are completely complementary to the target in this region will cleave. Can be performed. In a preferred embodiment, the mismatch occurs at a nucleotide position in the probe that hits the 5 ′ end of the site where cleavage occurs if there is no mismatch.

  In another embodiment, strictly require duplication (eg, using Afu FEN for DNA target detection or using 5 ′ nuclease of DNA polymerase for RNA target detection as described above) An INVADER assay can be performed under conditions to provide an alternative means of detecting one nucleotide or another sequence variation. In one embodiment, the probe is selected such that the position of the target base suspected of having a mutation is at the 5 ′ end of the region complementary to the target of the probe. The upstream INVADER oligonucleotide is positioned so as to produce a single base overlap. If the target and probe oligonucleotide are complementary at the base in question, an overlap is formed and cleavage occurs. However, if the target does not complement the probe at this position, the base in the probe becomes part of the non-complementary 5 'arm, so there is no overlap between the INVADER oligonucleotide and the probe oligonucleotide. , Cutting is suppressed.

  It is also conceivable to detect different sequences in one reaction. Probes specific for different sequences can be labeled separately. For example, the probes can have different dyes or other detectable components, different lengths, etc., or can make a difference in the net charge of the product after cleavage. When labeled differently in one of these methods, the contribution of each specific target sequence to the final product can be calculated. This can be applied when detecting the amount of different genes in the mixture. The different genes detected and quantified in the mixture may be wild type and mutant genes (eg, as found in cancer samples such as biopsies). In this embodiment, probes can be designed to be exactly the same site in some cases, but in other cases, one can be mutated to match the wild-type sequence. It can also be designed to match the type array. By quantitatively detecting the cleavage products obtained by the reaction that has been performed for a certain period of time, the ratio of the two types of genes in the mixture becomes clear. Such an analysis method is not limited to two types of genes. Many variants in the mixture can be measured as well.

  Alternatively, different sites of one gene can be observed and quantified, and the measured value of that gene can be confirmed. In this embodiment, the signal emanating from each probe is considered the same.

  In order to measure the signal of the aggregate, it is also conceivable to use a large number of probes without separately labeling them. This is desirable when using multiple probes designed to detect a gene in order to enhance the signal from that gene. This configuration can also be used to detect irrelevant sequences in the mixture. For example, when collecting blood, it is desirable to know if anyone is the host of an infectious agent. Regardless of which pathogen it is, blood will be discarded, so when applying the present invention to such a situation, the signals on the probe do not need to be separate and are actually confidential. Seems undesirable for.

  As explained above for the two-oligonucleotide system, the specificity of the detection reaction is affected by the length of the collection of target nucleic acid sequences involved in the hybridization of the entire set of detection oligonucleotides. For example, there may be applications where it is desirable to detect a single region in a complex genome. In such cases, a set of oligonucleotides that require accurate recognition can often be selected by hybridizing longer segments of the target nucleic acid, often 20-40 nucleotides. In another example, it may be desirable to have a set of oligonucleotides that interact with multiple sites in the target sample. In these cases, since one method is shorter, it seems statistically possible to use a set of oligonucleotides that recognize more general nucleic acid sequence segments.

  In one preferred embodiment, INVADER oligonucleotides and stacker oligonucleotides can be designed to be maximally stable so that they remain bound to the target sequence for an extended period of time during the reaction. This can be done by any of a number of methods well known to those skilled in the art, for example adding additional hybridizing sequences until the oligonucleotide is up to its length (up to about 50 nucleotides in total) or complementary strands. However, in order not to repel each other as much as a natural nucleic acid chain, it can be carried out by using a residue having a reduced negative charge such as a phosphorothioate or a peptide-nucleic acid residue. Such modifications also serve to make these neighboring oligonucleotides resistant to contaminating nucleases, thereby ensuring that they are more on the target strand during the course of the reaction. be able to. In addition, INVADER and stacker oligonucleotides can be covalently bound to the target (eg, by utilizing a psoralen bridge).

f) Application of Pooled DNA and RNA to Samples In some embodiments, the present invention measures pooled samples using INVADER detection reagents (eg, primary probes, INVADER probes, and FRET cassettes). Methods and kits are provided. In some preferred embodiments, the kit includes instructions on how to perform an INVADER assay, specifically from a sample collected from a number of individuals or from a single subject (eg, a biopsy sample). Instructions are provided on how the INVADER detection assay should be applied to “pooled” samples from the resulting large number of cells.

  In certain embodiments, the present invention provides a pooled sample collected from a large number of individuals (eg, 10, 50, 100, or 500 individuals) in a population, or a nucleic acid sequence derived from a large number of cells that are measured at one time. Makes it possible to detect polymorphisms in pooled samples collected from a single subject. In this regard, the present invention makes it possible to detect the frequency of rare mutations in the pooled sample and to confirm the allelic frequency of the population. In some embodiments, this allele frequency is then used to analyze the results of applying the INVADER detection assay to the individual's polymorphism frequency (eg, measured using the INVADER detection assay). Can do. In this regard, mutations that depend on the proportion of mutants found (eg, heterozygous loss mutations) can be analyzed and disease severity or disease progression can be determined (eg, given to Rapidus) U.S. Patent Nos. 6,146,828 and 6,203,993, which use genetic testing and statistical analysis to find mutations that cause disease or identify mutations that cause disease. In the case of identifying a patient sample to be included, it is incorporated herein by reference for all purposes).

  In some embodiments of the invention, extensive population screening is performed. In some embodiments, it is optimal to collect DNA from hundreds or thousands of individuals. In such a pool, for example, DNA from a single individual cannot be detected, and the detectable signal provides a measure of the frequency of alleles detected in a larger population. For example, the amount of DNA used is not determined by the number of individuals in the pool, but by the frequency of alleles detected. For example, in some embodiments, the assay produces a sufficient signal from 20-40 ng of DNA in a 90 minute reaction. This level of sensitivity corresponds to the resulting signal from alleles present in only about 3-5% of the population when analyzing 1 μg of DNA from a very complex pool.

g) Application of RNA detection method
RNA quantification is becoming increasingly important in basic research, pharmacy and clinical research. For example, disease progression and therapeutic effects can be predicted by quantifying viral RNA. Similarly, gene expression analysis of diseased vs. normal or untreated vs. treated tissues can identify relevant biological responses and evaluate drug effects. As the core of the Human Genome Project moves to gene expression analysis, the field has a versatile RNA analysis that can quantitatively monitor many types of selectively transcribed and / or processed RNA. Technology should be needed.

  As described above for multiple allele detection methods, the multiplex RNA INVADER assay allows for the expression analysis of two or more genes in the same sample. In a primary reaction, a single base overlap substrate is created by hybridizing INVADER oligonucleotide and probe oligonucleotide to their respective RNA targets. Each probe contains a specific target-complementary region and a characteristic non-complementary 5 'flap that binds only to the specific mRNA in the assay. Characteristic flaps can vary in size in any of the myriad methods disclosed herein (eg, different labels, different secondary cleavage systems with different labels, specific antibodies, when analyzed) And various sequences detected by hybridization in solution or on the surface).

  The RNA invasive cleavage assay can use two invasive cleavage reactions in the sequence (as described in Section II of the Detailed Description of the Invention below), similar to the method used for the DNA detection method described above. The selectivity for DNA polymerase-derived 5 ′ nucleases (detailed in Section IV of the Detailed Description of the Invention) indicates that it is preferable to change the mode further. Unlike the FEN 5 'nuclease commonly used to detect DNA targets, the signal from the DNA Pol-related 5' nuclease only when using the probe turnover mechanism in both the primary and secondary reactions Amplification occurs (as described in Section II of the Detailed Description of the Invention below, for example, metabolism of INVADER oligonucleotides with or without INVADER oligonucleotides to cleave multiple FRET cassettes. In contrast to the rotation mechanism). As a result, in a preferred embodiment, the RNA detection method uses a method in which two reactions are performed simultaneously rather than simultaneously. In these embodiments, the signal from the RNA INVADER assay accumulates linearly in a target-dependent and time-dependent manner so that the reaction is actually performed continuously. In contrast, the primary and secondary reactions of the DNA INVADER assay, when performed simultaneously, amplify the signal as a linear function of the target amount, but as a quadratic function of time. In a continuously performed embodiment, the RNA INVADER assay uses two separate oligonucleotides, a secondary probe (eg, a FRET probe), and a secondary target to generate a signal.

  An important feature of invasive RNA cleavage assays is the ability to distinguish highly homologous RNA sequences, such as those found in the cytochrome P450 gene family. Like the DNA INVADER assay, the RNA INVADER assay can distinguish single base changes. In some embodiments, the probe is cleaved because the first 5 ′ complementary base of each probe is located in a non-conserved site of its mRNA target and can form an overlapping structure if there is a mismatch To prevent it. By bringing the cleavage site into the splice site, the alternatively spliced mRNA variant can be specifically detected.

  Real-time analysis can be used to expand the dynamic range of the assay to monitor large changes in mRNA levels. However, since this assay generates a signal proportional to time or target amount, it is cheaper to calculate the number of copies of mRNA per 1 ng of total RNA by changing the amount of sample added per reaction. Accurate quantification is possible with a single end point measurement on a simple measuring instrument. In addition, if absolute quantification is not required, the linear signal amplification mechanism and reproducibility of this assay eliminates the need to create a standard curve and allows simple and accurate relative quantification of a single gene. To.

  RNA INVADER assays are particularly suitable for detecting alternatively spliced or edited RNA variants, since changes in the overlap site can affect 5 'nuclease cleavage even if they are single base changes . The technology can be used in all areas that require RNA quantification, such as high-throughput processing in drug discovery research, drug metabolism monitoring and clinical trial safety, and viral RNA clinical burden monitoring . According to this method, splice variants can be monitored in at least two ways. 1) detect each exon, or 2) detect a specific splice site.

  After splicing, use the INVADER assay probe set for each exon in question (or for all exons of mature RNA) to examine the RNA population for variants with more or fewer exons. design. Quantifying exons not only knows the expression level of each exon, regardless of how many mRNAs the exon is present in, but also gives information about the number of splice variants of that gene. For each probe, mini in vitro transcripts containing only one or a few exons can be made and absolute dosages can be made for each exon, allowing accurate comparison of exon amounts Become. When it is known that a particular exon is present in all known variants, in some embodiments, a probe for that exon is used for use as an internal control to average between different samples. Design the set. RNA with one copy of each exon (eg, a “normally spliced” RNA) is essential for a certain relative amount of signal from the set of probe sets (correcting for differences in the sensitivity of each probe set). Should be equal for all exons; see Section V). Splicing mutations change the relative amount of exons. For example, if all of the RNA produced did not have one of the normal exons, the signal for that exon dropped to zero, and if half of the RNA did not have that exon, The signal to exons falls to 50%. More complex combinations of splice variants and a mixture of differently spliced mRNAs give a more complex and informative profile. The detection is not limited to exons. RNA populations can usually be observed for the presence of intron sequences that are removed by splicing. Such extensive exon screening provides biologically relevant and diagnostic information when comparing normal versus diseased or untreated versus treated cells (eg, in pharmacogenomic screening assays). Is obtained. The description of this type of measurement result using an array is called an alternative splicing detection array method (D. Black, Cell 103: 367 [2000]). Similar results are obtained with the INVADER assay of mRNA, but with higher specificity and more accurate quantification results than the array method with oligonucleotides.

  In an alternative embodiment, alternatively spliced mRNA is detected by examining specific splice sites. The advantage of observing the splice site rather than the exon itself is that splice variants that contain very short exons (eg, less than 10 nucleotides) can be accurately detected by this assay.

  Further, in some embodiments, an mRNA INVADER assay is used to observe selective start and stop sites in the mRNA, as well as processed RNA and RNA fragments, and unprocessed RNA and Observe the lifetime of the RNA fragment (eg, used in time course experiments after induction).

II. Signal enhancement by incorporating products of invasive cleavage reactions into successive invasive cleavage reactions As described above, oligonucleotide products released by invasive cleavage can be subsequently converted to oligonucleotides within the size of the cleavage product. It can be used in the reaction or readout method used. In addition to the reactions involving primer extension and transcription described herein, another enzymatic reaction utilizing oligonucleotides is an invasive cleavage reaction. The present invention provides a method that uses an oligonucleotide released in a primary invasive cleavage reaction as a component to complete the cleavage structure and enable a secondary invasive cleavage reaction. The continuous use of products from invasive cutting is not limited to one additional step. It is also conceivable to carry out separate invasive cutting reactions successively.

  The polymerase chain reaction uses a DNA replication method that produces copies of the target segment of nucleic acid at a rate that accumulates exponentially. This is made possible by the fact that when the DNA strands are separated, each strand contains enough information to allow assembly of a new complementary strand. As new chains are synthesized, the number of identical molecules doubles. By repeating this process 20 times, the first strand can be replicated 1 million times, and very rare sequences can be easily detected. Increasing reactions at 2X mathematical magnification have been incorporated into many amplification assays.

By performing a number of consecutive invasive cleavage reactions, the method according to the present invention obtains the mathematical benefits of an exponent without producing extra copies of the target analyte. In a simple invasive cleavage reaction, the yield Y is simply the turnover rate K multiplied by the reaction time t (ie, Y = (K) (t)). When Y is used to represent the yield of a simple reaction, the yield of the compound (ie, a number of consecutive reactions) is assumed to be n consecutive, assuming that each invasive cleavage step has the same turnover rate. The number of invasive cleavage reactions performed can be simply expressed as Y ⁿ . If the yield in each step is different, the final yield is represented by a number calculated by multiplying the yield in each separate reaction of the continuous reaction. For example, if a primary invasive cleavage reaction can produce 1000 products in 30 minutes, and each of these products can in turn participate in 1000 additional reactions in turn, then in the second reaction, 1000 ² copies There will be (1000x1000) end product. If the third reaction is further performed in this series, the theoretical yield is 1000 ³ (1000 × 1000 × 1000). In the method according to the invention, the index is based on the number of invasive cleavage reactions in cascade. This is limited to 2 which is the number of strands in double-stranded DNA, and the index n is the number of cycles performed, so it requires multiple iterations to accumulate a large amount of product. This is a point that can be compared with an amplification method (for example, PCR).

  In order to distinguish the above exponential amplification method from the method according to the present invention, the former can be considered as an alternating reaction. This is because the reaction product is fed back to the same reaction (eg, event 1 results in multiple events 2 and each event 2 results in multiple events 1). In contrast, events in some embodiments of the invention are continuous (e.g., event 1 results in multiple events 2, each event 2 results in multiple events 3, etc. It does not contribute to the earlier event in the chain).

  Alternate method sensitivity is also one of the greatest weaknesses when these assays are used to determine the presence or absence of a target nucleic acid sequence in a sample. Since these reaction products are detectable copies of the starting material, contamination of the previous reaction product with the new reaction will produce false positive results (i.e., the target analyte will actually be The target nucleic acid appears to have been detected in the sample that does not contain any). In addition, the product concentration in each positive reaction is so high that enough DNA to generate a strong false positive signal can be combined with a new reaction very easily by contact with contaminated equipment or by aerosol. Can do. In contrast to the alternating method, the most concentrated product of the continuous reaction (ie, the product released in the final invasive cleavage reaction) will start a similar reaction or cascade when carried over to a new test sample It is not possible. This is in contrast to the exponential amplification method described above, because the reaction according to the invention can be carried out without expensive containment measures (eg special equipment or isolated laboratory space) that are required with the alternating method. This is a significant advantage. Inadvertent transfer of the product from the second reaction to the end results in the background of the final product in the absence of the target analyte, but a very large amount of contamination is sufficient to represent a risk of producing false positive results. I think things will be needed.

  When the term continuous is used, the present invention is arranged in such a way that one invasive cleavage reaction or assay must be completed before a subsequent reaction to invasively cleave another probe is initiated. It is not intended to be limiting. Rather, this term refers to the order of events that can occur if only one copy of each oligonucleotide species is used in the assay. The primary invasive cleavage reaction means which event occurs first in response to the formation of a cleavage structure on the target nucleic acid. Subsequent reactions, referred to as secondary reactions, tertiary reactions, etc., can only include an artificial “target” strand that serves only to assist in the assembly of the cleavage structure and is independent of the nucleic acid analyte of interest. . A complete assay modifies reaction conditions that allow subsequent cleavage reactions, such as local (eg, in separate reaction vessels) or temporal (eg, temperature, enzyme identity, or solution conditions), if desired. Can be configured with an isolated invasive cutting step, but all of the reaction components are mixed to initiate the secondary reaction as soon as the product produced by the primary cleavage is available It is possible to make it possible. In such a scheme, a primary cut, a secondary cut, and a subsequent cut involving copies of separate cut structures will occur simultaneously.

  It can be expected that there are several levels of this type of linear amplification method, where each successive reaction produces an oligonucleotide that can be involved in the cleavage of another probe in subsequent rounds. The primary reaction is specific to the analyte of interest, and secondary reactions (and tertiary reactions, etc.) are used to generate a signal while initiation is dependent on the primary reaction.

  The liberated product can be used for several possibilities in subsequent reactions. For example, the product of one invasive cleavage reaction becomes an INVADER oligonucleotide that specifically cleaves another probe in the second reaction. In such an example, the first invasive cleavage structure is formed by annealing the INVADER oligonucleotide and the probe oligonucleotide (probe 1) to the first target oligonucleotide (target 1). Depending on which part of the INVADER oligonucleotide and probe oligonucleotide can hybridize to the target, the target nucleic acid is divided into three regions. Target region 1 is complementary only to the INVADER oligonucleotide, target region 3 is complementary only to the probe, and target region 2 is between the INVADER oligonucleotide and the probe oligonucleotide. Have overlapping parts.

  Cleavage of probe 1 releases “cleavage probe 1”. The released probe 1 is then used as the INVADER oligonucleotide for the second cleavage. The second cleavage structure is formed by annealing the cleavage probe 1, the second probe oligonucleotide (“Probe 2”), and the second target nucleic acid (“Target 2”). In some embodiments, probe 2 and the second target nucleic acid are each preferably covalently linked at their 3 ′ and 5 ′ ends, thus forming a hairpin stem and loop, It is named. This loop may be a nucleic acid or a spacer or linker other than a nucleic acid. Inclusion of excess amounts of cassette molecules allows each of the cleavage probes 1 to act as an INVADER to cleave multiple copies of the cassette.

By labeling probe 2 and detecting the labeled cleavage probe 2, it can be detected that the second cleavage structure has been cleaved, but the label is a radioisotope (eg, ³² P or ³⁵ S ), Fluorophores (eg fluorescein), reactive groups detectable by secondary factors (eg biotin / streptavidin), positively charged adducts detectable by selective charge reversal (discussed in Section IV) Etc. Alternatively, cleavage probe 2 is either used in a tailing reaction, used to complete or activate protein binding sites, or any of the oligonucleotide detection or use methods described herein. Can be detected or used.

  In another embodiment, the probe oligonucleotide that is cleaved in the primary reaction is designed to fold back into its own shape (ie, self-complementary), so that it is a target oligonucleotide as an INVADER oligonucleotide A molecule (referred to as an “IT” complex) that can also act as The IT complex can then cleave another probe present in the secondary reaction. If the secondary probe molecule (“Probe 2”) is included in excess, each IT complex can act as a basis for generating multiple copies of the cleaved secondary probe. Depending on which part of the INVADER and probe oligonucleotides can hybridize to the target, the target nucleic acid is divided into three regions (as described above, but when a stacker oligonucleotide is used, the target Note that is divided into four regions). The second cleavage structure is formed by annealing the second probe (“Probe 2”) to a fragment of Probe 1 (“Cleaving Probe 1”) released by cleavage of the first cleavage structure. Since the self-complementary region contained in cleavage probe 1 anneals (this self-annealed cleavage probe 1 forms an IT complex), cleavage probe 1 has a hairpin or stem / loop structure 3 ′ Form at the end. The IT complex (cleavage probe 1) is divided into three regions. Region 1 of the IT complex is complementary to the 3 ′ site of probe 2, region 2 is complementary to both the 3 ′ end of cleavage probe 1 and the 5 ′ end of probe 2, and region 3 Has a self-complementary region (ie, region 3 is complementary to the 3 ′ site of cleavage probe 1). For the IT complex (ie, cleavage probe 1), region 1 is located upstream of region 2 and region 2 is located upstream of region 3. For another aspect of invasive cleavage, region “2” corresponds to a region where there is a physical overlap, not a sequence overlap, between the INVADER oligonucleotide site of cleavage probe 1 and the probe 2 oligonucleotide. There is.

  A complex incorporating an INVADER-target that detects the cleavage product of a secondary invasive cleavage reaction (ie, cleavage probe 2) or is used in turn with a third probe molecule, It can be designed to constitute a complex independent of the target.

  The oligonucleotide product of the primary cleavage reaction can serve any of the oligonucleotides described herein (e.g., can act as a target without a binding INVADER oligonucleotide-like sequence, or It can also act as a stacker oligonucleotide as described above), and by stabilizing probe hybridization by coaxial stacking, the turnover rate seen in secondary reactions can be increased.

  The secondary cleavage reaction in some embodiments of the invention includes a FRET cassette. Such molecules provide a secondary target and a FRET-labeled cleavable sequence to detect a continuous invasive cleavage reaction homogeneously (i.e. without separation of the product or other manipulation after the reaction). Detection). In another preferred embodiment, a secondary reaction system is used in which the FRET probe and the synthetic target are provided as separate oligonucleotides.

  In a preferred embodiment, each subsequent reaction is facilitated by (ie, dependent on) its previous cleavage product so that the presence of the final product is an indicator of the presence of the target analyte. However, the cleavage in the secondary reaction need not depend on the presence of the primary cleavage reaction product, as long as the primary cleavage reaction product can simply promote the rate of the secondary cleavage reaction in a measurable manner.

  In summary, the INVADER assay cascade (ie, continuous invasive cleavage reaction) according to the present invention is a combination of two or more linear assays that allow the final product to accumulate at an exponential rate. Yes, but there is no significant risk of carry-over contamination. Background that is not caused by continuous cleavage, such as secondary probe pyrolysis, generally increases linearly over time. In contrast, signal generation from a two-step continuous reaction follows quadratic dynamics. Therefore, time was elapsed by collecting data over time using an instrument that allows the determination of time points or real-time detection while incubating with the INVADER assay reaction method. An attractive ability to distinguish between true signal and background is provided based only on whether the signal sometimes increases quadratically or linearly. For example, in the figure, the true signal is represented as a quadratic curve and the background accumulation is a straight line, so it is easy even if the absolute amount of background signal (eg fluorescence in FRET detection) is quite large. Can be distinguished.

  The continuous invasive cleavage amplification method according to the present invention includes the detection method described herein (for example, analysis using a gel by standard methods or charge reversal), addition reaction by polymerase, protein binding region Can be used as an intermediate boost to the uptake reaction. When used in such a combination, increasing the production of specific cleavage products in an invasive cleavage assay reduces the sensitivity and specificity burden on the readout system.

  In addition to allowing various detection platforms, the cascade method is suitable for performing multiplex analysis of each analyte (ie, each target nucleic acid) in a single reaction. Multiplexing methods can be classified into two types. In one type, it is desirable to know the identity (and amount) of each analyte that can be present in a clinical sample, or the identity of each analyte and internal control. Several separate secondary amplification systems can be included to identify the presence of multiple analytes in a single sample. Each probe that is cleaved in response to the presence of a specific target sequence (or internal control) is linked to various detectable components, such as various sequences extended by DNA polymerase, or various dyes in the FRET format. Another cascade can be designed to start. This makes it possible to calculate the contribution of each specific target sequence to the final product to quantitatively detect various genes or alleles in a sample containing a mixture of genes or alleles.

  In the second configuration, it is desirable to determine which of several analytes are present in the sample, but the exact identity of each analyte need not be known. For example, when collecting blood, it is desirable to know if the blood of someone who is the host of an infectious agent is present in the blood sample. Regardless of which pathogen it is, blood will be discarded, so when applying the present invention to such a situation, the signals on the probe do not have to be separate and are actually confidential. Seems undesirable for. In this case, the 5 ′ arms of various analyte specific probes (ie, the 5 ′ sites released by cleavage) are identical, thus initiating the same secondary signal cascade. By using a similar configuration, use multiple probes complementary to one gene to enhance the signal from that gene or ensure that it is included when there are many allelic loci for the gene to be detected. Can do.

  There are two possible causes of background in the primary INVADER assay reaction. The first cause arises from INVADER oligonucleotide-independent cleavage of the probe annealing to the target, itself, or one of the other oligonucleotides present in the reaction. For this reason, it is preferable to use an enzyme that cannot efficiently cleave a structure without a primer. The enzyme Pfu FEN-1 does not cause detectable cleavage if no upstream oligonucleotide is present or if there is only an upstream oligonucleotide that cannot invade the probe-target complex. This indicates that Pfu's FEN-1 nuclease is suitable for use in the method according to the present invention.

  Other structure-specific nucleases are also suitable enzymes for use in the method according to the invention. As discussed in the first example, some 5 'nucleases can be used under conditions that significantly reduce this primer-independent cleavage. For example, when a hairpin is cleaved using a 5 'nuclease of DNAP Taq, a monovalent salt enters the reaction solution, and primer-independent cleavage is significantly reduced (Lyamichev et al., [1993], supra).

III. Effect of ARRESTOR oligonucleotides on signal and background in continuous invasive cleavage reactions As mentioned above, the higher the probe concentration, the higher the final signal yield. Can be raised. However, if a large amount of probe remains uncut, there will be a problem in the subsequent detection or further amplification using the cleavage product. If the subsequent steps are simple detection (eg, detection by gel analysis), excess uncut material may streak or disperse the signal or overwhelm the detection molecules ( For example, in the case of radioactivity, it may cause background to occur (eg, overexposed film, exceeding the quantitative detection limit of the fluorescence imaging device). This problem can be solved by partitioning the product from the uncleaved probe. In more complex detection methods, the cleaved product can interact with another substance to indicate cleavage. As described above, the cleavage products can be used in reactions that use oligonucleotides, such as hybridization, primer extension reactions, ligations, or invasive cleavage instructions. In any of these cases, it is necessary to consider what to do with the remaining uncleaved probe when planning the reaction. In the primer extension reaction, the uncleaved probe may hybridize to the template and extend. In order to elongate, the hybridized uncleaved probe will not elongate if it is necessary to cleave to produce the correct 3 'end. However, it competes with the cleavage product for the template. If the template is present in excess of the combined cleaved and uncleaved probes, it should be possible to find a template copy for binding of both probes. However, if there is only a limited amount of template, competition will reduce the percentage of cleavage probes that can successfully bind to available templates. If a large excess of probe was used to trigger the initiation reaction, the remaining probe would also be present in excess in excess of the cleavage product, making it an effective competitor and the final detection reaction This reduces the amount of product (eg, extension reaction product).

  When uncleaved probe materials are involved in secondary reactions, they can cause background in these reactions. Presenting a cleaved probe for subsequent reactions can be an ideal substrate for the enzyme used in the next step, but depending on the enzyme, it may be less efficient or even an uncleaved probe. There is a possibility of acting. In the course of developing the present invention, it was found that even if one of the nicked promoters has additional unpaired nucleotides, transcription from such promoter is promoted. Similarly, when the subsequent reaction is an invasive cut, the non-cutting probe may bind to a component for forming a second cutting structure with the cutting probe. In experiments conducted in the course of developing the present invention, it was found that the 5 ′ nucleases described herein can catalyze any treatment that cleaves the defective structure. Even at low amounts, this aberrant cleavage may be misinterpreted as a positive target-specific cleavage signal.

  Given these negative effects of having an excess of uncleaved probes, there is clearly a need for some way to prevent such interactions. As described above, after the primary reaction, it is possible to isolate the cleavage product from the non-cleavable probe by a conventional method. However, these methods are often time consuming and expensive (eg, disposable columns and gels) and can increase the risk of sample manipulation and contamination. It is more preferable to design the continuous reaction so that it does not require transfer of the first sample to a new container for subsequent reactions.

  The present invention provides a method for reducing the interaction between the primary probe and other reactants. The method provides a method that specifically avoids uncleaved probes from participating in subsequent reactions. Such avoidance can be achieved by incorporating into the next reaction step an agent designed to specifically interact with the uncleaved primary probe. Considering the primary probe in an invasive cleavage reaction for convenience reasons, ARRESTOR molecules can be used in the reaction step in a series of invasive cleavage steps when necessary or desirable for assay design. The ARRESTOR molecule according to the present invention is not limited to a specific process.

  A method to avoid remaining uncleaved probes from the primary reaction utilizes factors that can be specifically designed or selected to bind to uncleaved probes with greater affinity than cleaved probes, thereby Allows the cleaved probe molecular species to efficiently compete for components of subsequent reactions, even in the presence of a large excess. These factors are termed “ARRESTOR molecules” because they have the ability to stop the primary probe from participating in subsequent reactions. In the various examples described below, oligonucleotides are provided as ARRESTOR molecules in invasive cleavage assays. Molecules or chemical agents that can distinguish between full-length uncleaved probes and cleaved probes and selectively bind or otherwise disable uncleaved probes are within the meaning of the present invention. It is thought that it can act as an ARRESTOR molecule. For example, selecting through a number of in vitro amplification steps (eg, “SELEX”, US Pat. Nos. 5,270,163 and 5,567,588, which are incorporated herein by reference), and a specific number of capture or other selection means Antibodies with such specificity can be obtained so that possible “aptamers” are obtained.

  In one embodiment, the ARRESTOR molecule is an oligonucleotide. In another embodiment, the ARRESTOR oligonucleotide is a composite oligonucleotide that is not covalently linked but binds cooperatively and is stabilized by coaxial stacking. including. In a preferred embodiment, the oligonucleotide is modified to reduce interaction with the cleavage factor according to the present invention. When an oligonucleotide is used as an ARRESTOR oligonucleotide, it is intended not to participate in subsequent reaction steps. When the ARRESTOR oligonucleotide binds to the primary probe, the secondary target becomes a substrate that is cleaved by the 5 ′ nuclease used in some embodiments of the invention, with or without secondary involvement. A branch structure can be formed. Formation of such a structure may result in some undesired cleavage that contributes to the background, reduces the specific signal, or causes competition for the enzyme. It is preferable to provide an ARRESTOR oligonucleotide that does not form such a cleavage structure. One way to do this is because the INVADER oligonucleotide occupies a similar position in the cleavage structure (ie, the 3 'end of the INVADER oligonucleotide is at the unpaired 5' arm cleavage site). The modification of the oligonucleotide is known to reduce the activity of the INVADER oligonucleotide. In the following Example section, when the influence of modification of the 3 ′ end of the INVADER oligonucleotide on cleavage was examined, it was clearly found that the action of the INVADER oligonucleotide was weakened. Other modifications not described herein can also be easily characterized by performing tests with the cleavage enzyme used in the reaction intended for the ARRESTOR oligonucleotide.

  In preferred embodiments, the backbone of the ARRESTOR oligonucleotide is modified. Doing this increases resistance to degradation by nucleases or temperature, resulting in a double-stranded structure (eg, Type A duplex vs. Type B duplex) that is unsuitable for the enzyme used. In particularly preferred embodiments, the modification comprises a 2′O-methyl substitution in the backbone of the nucleic acid, but in particularly preferred embodiments, the 2′O-methylated modified oligonucleotide further comprises a 3 ′ terminal amine group.

  The purpose of the ARRESTOR oligonucleotide is to allow a small population of cleaved probes to compete with a non-cleaved probe for binding, regardless of what component is used in the next step. ARRESTOR oligonucleotides that can completely distinguish between the two probe species (eg, bind only to non-cleavable and not to non-cleavable) are most beneficial in some embodiments, In many applications, such as the continuous INVADER assay described herein, the ARRESTOR oligonucleotides according to the present invention have only a partial discriminating ability, but may perform as intended. it can. When ARRESTOR oligonucleotides interact with cleaved probes, they can prevent detection of certain sites of these cleavage products, which can reduce the amount of absolute signal generated from a given amount of target substance. . If this same ARRESTOR oligonucleotide has the effect of reducing the background of the reaction (ie, non-target specific cleavage) by a factor that is greater than the factor by which the specific signal is reduced, the absolute amount of signal is reduced. Even so, the significance of the signal (ie the ratio of signal to background) increases. By comparing the amount of background and specific signal generated from a reaction that does not contain an ARRESTOR molecule with the amount of background and specific signal generated from a reaction that contains an ARRESTOR molecule, the possible design of the ARRESTOR molecule Can be examined in a simple way. What constitutes an acceptable exchange level of an absolute signal for specificity differs for different applications (eg, target amount, read sensitivity, etc.) and is thus determined by the user using the method according to the invention. be able to.

IV. An improved enzyme cleavage structure for use in INVADER oligonucleotide-specific cleavage reactions involving RNA targets is defined herein as a structure formed by the interaction of a target oligonucleotide with a probe oligonucleotide that forms a duplex. The resulting structure can be cleaved by a cleaving factor such as, but not limited to, an enzyme. A cleavage structure is further defined as a substrate for specific cleavage by a cleavage means, as opposed to a nucleic acid molecule that is a substrate for non-specific cleavage by factors such as phosphodiesterase. In considering an improvement to an enzymatic cleavage factor, the action of the enzyme on a structure that falls within the definition of a cleavage structure can be considered. Specific cleavage at sites within such structures is possible.

  Enzyme improvements may increase or decrease the rate at which one or more structures are cleaved. Improvements may also result in increased or decreased cleavage sites in one or more of the cleavage structures. In developing a library of new structure-specific nucleases for use in nucleic acid cleavage assays, the improvements have many aspects, each related to the specific substrate structure used in a particular assay.

  As an example, one embodiment of the INVADER oligonucleotide-specific cleavage assay method according to the present invention can be considered. In the INVADER oligonucleotide specific cleavage assay, the accumulation of cleaved material is affected by several characteristics of the enzyme behavior. Not surprisingly, the turnover rate, or the number of structures that can be cleaved by a single enzyme within a certain time, is very important in determining the amount of substance processed in the measurement reaction. . If the enzyme takes a long time to recognize the substrate (eg if the substrate is presented in a non-optimal structure) or if it takes time to cleave, the product accumulates The speed will be lower than when those processes proceeded quickly. Even if these steps are rapid, if the enzyme is “hold on” on the cleavage structure and cannot immediately proceed to another non-cleavage structure, it will have a negative impact on the rate.

  Only the rate of enzyme turnover does not negatively influence the behavior of the enzyme on the rate of product accumulation. If the method used to visualize or measure the product is specific to a precisely defined product, the product will not be detected so that the product accumulation rate is It may seem to be lower. For example, if the detection device has a high sensitivity for detecting trinucleotides and does not detect dinucleotides, tetranucleotides, or oligonucleotides other than three residues, the INVADER-specific In a selective cleavage assay, incorrect cleavage will result in a proportional decrease in detectable signal. As can be seen from the cleavage data presented here, it is often possible to cleave at least one nucleotide away from the primary cleavage site, even if there is only one site in the probe that is suitable for cleavage. Product is present. These are target-dependent and non-specific background products. Nevertheless, if the subsequent visualization system can only detect the primary product, these mean the disappearance of the signal. An example of such a selective visualization system is the charge reversal reader presented here, where the balance of positive and negative charges determines the behavior of the product. In such a system, if there are extra nucleotides or no expected nucleotides, legitimate cleavage products can be excluded from final detection by leaving the product with the wrong charge balance. Assays that can sensitively distinguish the nucleotide content of oligonucleotides, such as standard stringent hybridization, can cause sensitivity problems when a fraction of legitimate products cannot be successfully detected by the assay Can be easily understood.

  These considerations suggest two highly desirable traits in the enzyme to be used in the method according to the invention. First, the faster the enzyme performs all cleavage reactions such as recognition, cleavage and release, the more likely it is that a stronger signal is generated in the INVADER oligonucleotide-specific cleavage assay. Second, the more concentrated the enzyme is at a single cleavage site in the structure, the more successful it can be in detecting more cleavage products in the selective readout method.

  The principles quoted above for improving the enzymes used in INVADER oligonucleotide-specific cleavage assays are intended to be used as an example to show one direction in which improvements are sought, and improved enzyme activity. It is not intended to limit either the nature of or the application. As another example of a change in activity as considered to be sufficiently improved, a DNAP-binding 5 ′ nuclease can be used as an example. In creating some of the polymerase-deficient 5 'nucleases described herein, it was discovered that an enzyme made by deleting a substantial portion of the polymerase domain had weak or non-existing activity in the original protein. . These activities include the ability to cleave unforked structures, very high ability to exonucleolytically remove nucleotides from the 5 'end of the duplex, and even without a free 5' end It included an incomplete ability to cleave cyclic molecules.

In addition to 5 ′ nucleases derived from DNA polymerases, the present invention also contemplates the use of structure-specific nucleases that are not derived from DNA polymerases. For example, a class of eukaryotic and archaeal endonucleases have been identified that have a substrate specificity similar to the 5 'nuclease of Pol type I DNA polymerase. These FEN1 (Flap endonuclease: F lap E ndo N uclease) , RAD2, and XPG (Xeroderma pigmentosum group G) proteins. These proteins are implicated in DNA repair and have been shown to prefer to cleave structures resembling 5 'arms displaced by primers that extend during the polymerization process. Similar DNA repair enzymes have been isolated from single cells and higher eukaryotes and archaea, and there are DNA repair proteins associated with eubacteria. Similar 5 'nucleases were also associated with bacteriophages such as T5 and T7.

  Recently, the three-dimensional structure of 5'-exonucleases of DNAPTaq and T5 phage has been determined by X-ray diffraction (Kim et al., Nature 376: 612 [1995]; and Ceska et al., Nature 382: 90 [1995]) . These two enzymes have very similar three-dimensional structures despite limited amino acid sequence similarity. The most prominent features of T5 5'-exonuclease are the presence of a triangular hole formed by the active site of the protein and the presence of two alpha helices. The same region of DNAPTaq is disturbed in the crystal structure, indicating that this region is plastic. For this reason, this region is not shown in the published three-dimensional structure. However, based on the overall three-dimensional structural similarity to T5 5'-exonuclease and the fact that the amino acids in the disordered region of the DNAPTaq protein are those involved in alpha helix formation, the 5 'nuclease of DNAPTaq Domains are likely to have the same structure. The presence of such a hole or groove in the 5 'nuclease domain of DNAPTaq was predicted based on substrate specificity (Lyamichev et al., Supra).

  It has been suggested that in order for the structure to be properly cut, the 5 'arm of the cutting structure must pass through the helical arch described above (Ceska et al., Supra). One modification of the 5 'nuclease described herein is to open this helical arch site in the protein so that structures that can hardly be cleaved or cannot be cleaved at all can be better cleaved (eg, Such a structure on a circular DNA target that can exclude the passage of the 5 'arm). The genetic construct chosen as a model for testing this method was a construct derived from DNAPTaq called CLEAVASE BN, but without the polymerase domain. This should be very close in structure to the T5 5'-exonuclease because it contains all of the 5 'nuclease domain of DNAPTaq. This 5 'nuclease was chosen to demonstrate the principle of such physical modification for this type of protein. The arch-opening modification according to the present invention should not be limited to the 5 ′ nuclease domain of DNA polymerase, but is intended to be used for structure-specific nucleases containing an opening that may be a restriction on cleavage activity. Has been. The present invention envisages inserting thrombin cleavage sites into the helical arch of DNAP from Thermus and into the helical arch of the 5 ′ nuclease from Thermus. The specific example shown here using CLEAVASE BN / thrombin nuclease only illustrates the concept of opening the helical arch present inside the nuclease domain. Since the amino acid sequences of DNAPs from Thermus are highly conserved, in accordance with the teachings of the present invention, thrombin is incorporated into these DNAPs and the helical arches present in 5 'nucleases derived from these DNAPs. It becomes possible to insert a site.

  The spiral arch was opened by inserting a protease site into the arch. This allowed post-translational degradation of the expressed protein with a protease suitable for opening the arch at its tip. This type of protease recognizes short linear chains with specific amino acid sequences. Such proteases include thrombin and factor Xa. Such cleavage of a protein by a protease is determined by the presence of the site in the amino acid sequence of the protein and the ability to contact the site on the folded original protein. Even using crystal structures, it can be difficult to predict that certain regions of a protein are sensitive to cleavage by proteases. If there is no crystal structure, it must be determined empirically.

  In selecting a protease that site-specifically cleaves a protein that has been modified to include a protease cleavage site, the first step is to examine the unmodified protein for cleavage at another site. For example, DNAPTaq and CLEAVASE BN nuclease were incubated under protease cleavage conditions with factor Xa protease and thrombin protease. Both nucleotide proteins were cleaved by factor Xa within the 5 ′ nuclease domain, but neither nuclease was degraded by large amounts of thrombin. Therefore, thrombin was first selected to examine the arch opening of the CLEAVASE BN enzyme.

  In the protease / CLEAVASE modification described herein, factor Xa protease was cleaved strongly at unacceptable sites in the unmodified nuclease protein, ie, areas that are likely to compromise the activity of the final product. Other unmodified nucleases contemplated herein are not sensitive to factor Xa, but may be sensitive to thrombin and other similar proteases. Alternatively, it may be sensitive to these proteases, or other similar proteases, at sites that are not critical for nuclease function in need of modification. When picking up a protein for modification by adding a protease cleavage site, the unmodified protein is used as a protease to determine which protease produces an acceptable level of cleavage in another region. Should be tested.

  Using a cloned DNAPTaq segment in which the CLEAVASE BN protein is expressed, a nucleotide encoding the thrombin cleavage site was introduced in-frame near the sequence encoding the 90th amino acid of the nuclease gene. This position was determined to be at or near the tip of the helical arch with reference to both the three-dimensional structure of DNAPTaq and the structure of T5 5 ′ exonuclease. The encoded amino acid sequence LVPRGS was inserted at the tip of the helical arch by site-directed mutagenesis of the nuclease gene. The proline (P) at the thrombin cleavage site was placed to replace the proline normally in this position with CLEAVASE BN. Because it is an amino acid that destroys the α helix, it may be important for the three-dimensional structure of this arch. This construct was expressed, purified and then cleaved with thrombin. It was examined whether the degraded enzyme can cleave DNA that does not have a free 5 ′ end to promote cleavage by a threading model with the genomic DNA of the target nucleic acid bacteriophage M13.

  Although this nuclease helical arch was opened by protease cleavage, it is believed that many other techniques can be used to accomplish the same purpose. For example, when the base sequence is rearranged and expressed, the resulting protein has a helical arch tip (amino acid at position 90) that is the amino terminus of the protein and the original carboxyl terminus of the protein sequence. The amino terminus would be attached and the new carboxyl terminus would be designed to be the original 89th amino acid. This method has the advantage that it does not introduce foreign sequences and the enzyme is a single amino acid chain and therefore will be more stable than a cleaved 5 'nuclease. In the crystal structure of DNAPTaq, the carboxyl terminus and amino terminus of the 5 'exonuclease domain are very close to each other, and these ends can be directly joined, sometimes without using the necessary plastic linker peptide sequence. It suggests. Such gene rearrangements can be performed by standard PCR recombination methods, as well as subsequent cloning and expression, and by cloning techniques known to those skilled in the art.

The INVADER invasive cleavage reaction has been shown to be useful for detecting RNA target strands (see, eg, US Pat. No. 6.001,567, which is hereby incorporated by reference in its entirety). Incorporated into the book). Conditions that can cleave many copies of the probe for each copy of the target RNA present in the reaction, as was the case with the INVADER assay for DNA detection (Lyamichev et al., Nat. Biotechnol., 17: 292 [1999]) The reaction can be carried out under In one embodiment, the reaction can be carried out at a temperature close to the melting temperature (T _m ) of the cleaved probe so that the cleaved and non-cleaved probes do not change temperature periodically. Can easily and repeatedly attach to and detach from the target strand. The full-length probe is cleaved by the 5 ′ nuclease each time it binds to the target in the presence of the INVADER oligonucleotide, resulting in the accumulation of the cleavage product. This accumulation is highly specific to the sequence to be detected and can be designed to be proportional to the reaction time and target concentration in the reaction. In another embodiment, the reaction temperature is changed (ie, can be raised to a temperature at which the probe can be dissociated) and then to a temperature at which a new probe copy hybridizes to the target and is cleaved by the enzyme. Can be reduced. In a further embodiment, similar to PCR, the process of raising and lowering temperature is repeated many times or periodically (Mullis and Faloona, Methods in Enzymology, 155: 335 [1987], Saiki et al., Science 230: 1350 [1985]).

  As described above, the 5 ′ nuclease of Pol A type DNA polymerase is suitable for cleaving an invasive cleavage structure containing an RNA target strand. The present invention provides enzymes that exhibit improved efficiency in detection methods that utilize cleavage of RNA-containing structures. In particular, the modified polymerase according to the present invention exhibits improved efficiency in a detection method that utilizes cleavage of a DNA portion of an invasive cleavage structure containing an RNA target strand.

  The 5 ′ nuclease according to the present invention can be derived from Pol A type DNA polymerase. The terms used in describing modifications to this type of 5 'nuclease correspond to those described in the structure of DNA polymerases known in the art. The Klenow fragment of Pol A DNA polymerase derived from E. coli (the C-terminal two-thirds region, which has DNA synthesis activity but no 5 'nuclease activity) has a physical shape similar to that of the right hand. It is explained to have. That is, an open region called “palm” and a cleft holding a primer / template duplex, one defined by the “finger” domain and the other defined by the “sum” domain. (Joyce and Steitz, Trends in Biochemical Science 12: 228 [1987]). This is shown in FIG. Since this physical form has proven to be common for many other template-dependent polymerases such as Pol A-type DNA polymerase and reverse transcriptase, manual terminology is used in the art. As it becomes known, the active site in these enzymes is often described corresponding to the position of the hand. Not intended to limit the present invention, but for reference, palm is made from almost the first 200 amino acids of the polymerase domain, thumb is made from the central 140 amino acids, and the fingers are It consists of 160 amino acids, and the base of the cleft is formed from the remaining region (Fig. 6). Some enzymes deviate from the description of these structures, but it is possible to identify equivalent domains and sequences corresponding to these domains by methods such as sequence homology to known enzyme sequences or by comparing enzyme crystal structures. it can.

  Although the present invention is not limited to a specific method, several methods are used in preparing the improved enzyme according to the present invention. First, two DNA polymerases, Taq and Tth, with different rates of DNA strand breakage activity on RNA targets were compared. In order to identify domains involved in activity differences, a series of chemical constructs were created and their activity was measured. In this step, two regions of Tth polymerase were identified that, when transferred to Taq polymerase, can confer RNA-dependent cleavage activity equivalent to that of native Tth protein to TaqPol. As these regions were identified, the specific amino acids involved in this activity were examined. The two proteins were approximately 87% identical in overall amino acid sequence, and the identified regions differed by only a few amino acids. By changing one or more of these amino acids one by one or more, TthPol has identified amino acid pairs that, when introduced into TaqPol protein, increase the cleavage rate to that of natural TthPol.

  These data reveal two important aspects of the present invention. One is that TaqPol-like RNA-dependent cleavage activity can be imparted to a polymerase with lower activity by changing specific amino acids. However, in a broader sense, these results provide regions of these polymerases that are involved in the recognition of cleavage structures including RNA. With the identification of these important regions, public information on the relationship of other amino acids to the various functions of these DNA polymerases and the computational molecular modeling performed during the development of the present invention Together, it allowed a rational design method to create a further improved 5 'nuclease. This information also enabled a focused random mutagenesis coupled with a rapid screening method to quickly create and identify enzymes with improved properties. A variety of improved polymerases are provided using these methods according to the present invention.

  Methods useful in creating and selecting improved 5 ′ nucleases according to the present invention are detailed below and in the Examples. General procedures for screening and characterizing 5 ′ nuclease cleavage activity are included in the examples. Method considerations are divided into the following items: I) Creation and selection of chimeric constructs; II) Site-directed mutagenesis based on information from chimeric constructs; III) Site-directed mutagenesis using molecular modeling and published physical studies; IV) Directed randomization Mutagenesis

1) Creation and selection of chimeric constructs As illustrated in Figure 6, Pol A DNA polymerases, such as DNA polymerase enzymes derived from Thermus species, contain two distinct domains: a 5 'nuclease domain and a polymerase domain . The polymerase domain is present in the C-terminal two-thirds region of this protein and is involved in DNA-dependent and RNA-dependent DNA polymerase activities. The N-terminal third part contains a 5 'nuclease domain. In Thermus PolA polymerase, the palm region contains approximately amino acids at positions 300-500, the thumb region contains amino acids at positions 500-650, while the finger region is formed from the remaining amino acids at positions 650-830. (Fig. 6).

  Taq DN RX HT and Tth DN RX HT derived from Taq and Tth Po1, which are used in many of the experiments of the present invention and are also described herein, have reduced synthetic activity and facilitate the construction of chimeras. Have essentially the same 5 ′ nuclease activity as unmodified Taq Pol and Tth Pol. Unless otherwise noted, Taq Pol enzyme and Tth Pol enzyme in the following discussion mean DN RX HT derivatives.

  Taq Pol and Tth Pol show similar cleavage for the DNA target structure (Figure 10), but with the IL-6 RNA template (shown in Figure 10), Tth Pol has a cleavage rate 4 times faster than Taq Pol. (Shown in FIGS. 11 and 12). The Taq Pol and Tth Pol amino acid sequences (FIGS. 8 and 9) are approximately 87% identical and exhibit more than 92% similarity, so Taq Pol is highly homologous between these enzymes. A series of chimeric enzymes can be made between and Tth Pol. The activity of the chimeric enzyme was used as a parameter to identify regions of these proteins that affect RNA-dependent 5 'nuclease activity.

  The chimeric construct between the Taq Pol gene and the Tth Pol gene illustrated in FIGS. 7 and 19 was made by exchanging DNA fragments defined by the following restriction endonuclease sites: common to both genes EcoRI and BamHI, the cloning vector site SalI, and NotI, BstBI and NdeI created at site homologous to both genes by site-directed mutagenesis. Restriction enzymes are abbreviated as follows: EcoRI is E, NotI is N, BstBI is Bs, NdeI is D, BamHI is B, and SalI is S.

  The activity of each chimeric enzyme was assessed using an invasive signal amplification assay with IL-6 RNA target (FIG. 10) and the repeated cleavage rate shown in FIG. 12 was measured as described in the Examples. A comparison of the first two chimeric Taq Tth (N) and Tth Taq (N) (Figure 7), created by exchanging the polymerase and 5 'nuclease domains at the NotI site, shows that Taq Tth (N) While having the same activity as Pol, its counterpart, Tth Taq (N), retains Taq Pol activity (FIG. 12). This result indicates that the rate of repeated cleavage of Tth Pol is related to its polymerase domain, suggesting that the polymerase domain has an important role in 5 ′ nuclease activity.

  The next step was to identify the minimal region of Tth Pol polymerase that, when replaced with the corresponding Taq Pol sequence, can produce Tth Pol-like RNA-independent 5 'nuclease activity. To this end, a Taq Tth (N) chimera was selected and its Tth Pol sequence was replaced with a Taq Pol homology region, creating a series of new constructs. First, using the common BamHI site as a breakpoint to create Taq Tth (NB) and Taq Tth (BS) chimeras (Figure 7), the corresponding region of Taq Tth (N) was used as the Taq Pol polymerase domain. Replaced with the N-terminal part and C-terminal part of. Taq Tth (N-B) having an amino acid sequence from 328 to 593 of Tth Pol is about 3 times as active as Taq Tth (B-S), and 40% more active than Tth Pol (FIG. 12). As a result, it is confirmed that the NotI-BamHI site of the polymerase domain of Tth Pol determines the excellent RNA-dependent 5 ′ nuclease activity of Tth Pol.

  From these data, when the central part of Tth Pol was used to replace the Taq Pol homologous site (Taq Tth (NB) construct), the RNA recognition ability superior to the chimera protein composed mainly of Taq protein It was determined that can be provided. In fact, the rotational speed of this chimeric protein exceeded that of its parent Tth Pol. Comparing chimeras containing part of the increased activity of Tth Pol, in each case approximately 50% of the regions (Taq Tth (ND) and Taq Tth (DB), Figures 7 and 12) Comparison did not show a significant improvement in RNA-dependent activity. This result indicates that half of each feature of this region is required for this activity. Constructs with a portion slightly smaller than the Tth insertion (Taq Tth (Bs-B)) showed activity close to that of the parent Tth Pol protein, but less than that of the Taq Tth (NB) construct. It was.

2) Site-directed mutagenesis based on information from chimeric constructs
Comparison of Taq Pol and Tth Pol amino acid sequences between the BstBI and BamHI sites revealed that only 25 residues were different (FIG. 13). Of these, 12 are conservative changes and 13 substitutions cause changes in charge. Analysis of the chimeric enzyme suggested that there were several important amino acid mutations in the BstBI-NdeI and NdeI-BamHI regions of Tth Pol, and therefore, using site-directed mutagenesis, Taq Tth ( DB) and Taq Tth (ND) chimeras introduced Tth Pol specific amino acids into the BstBI-NdeI region and NdeI-BamHI region, respectively. Six Tth Pol specific substitutions were made in the BstBI-NdeI region of Taq Tth (DB) by single or double mutagenesis, and only one double mutant W417L / G418K was IL-6 It was possible to restore Tth Pol activity by the RNA target (see, for example, FIG. 14). Similarly, when 12 Tth Pol-specific amino acids were introduced at homologous positions in the NdeI-BamHI region of Taq Tth (ND), only one of them (E507Q) was comparable to Tth Pol. The cutting speed increased to (for example, see FIG. 14).

  In order to confirm that the substitutions W417L, G418K and E507Q are sufficient to raise the activity of Taq Pol to the level of Tth Pol, we made variants of Taq Pol with these mutations to create IL- Using 6 RNA substrates, their cleavage rates were compared with those of Tth Pol. Figure 15 shows that the Taq Pol W417L / G418K / E507Q and Taq Pol G418K / E507Q mutants have 1.4 times higher activity than Tth Pol and 4 times higher activity than Taq Pol, but Taq Pol W417L / The E507Q mutant has the same activity as Tth Pol, indicating that it is about 3 times higher than Taq Pol. These results demonstrate that Tth Pol's K418 and Q507 are important amino acids that define superior RNA-dependent 5 'nuclease activity compared to Taq Pol.

  By introducing mutations corresponding to the polymerase A gene of yet another organism, Thermus filiformis, and Thermus scottodactus, these amino acids can improve the RNA-dependent 5 'nuclease activity of DNA polymerase. Were tested. Taq Pol was modified to have the W417L and E507Q mutations and more closely resembled the corresponding residues of Tth Pol (K418 and Q507) and showed improved RNA-dependent activity. Tfi Pol was modified to have P420K and E507Q to create TfiDN2M, and TscPol was modified to have E416K and E505Q to create TscDN2M. The activity of these enzymes to cleave various structures including DNA and RNA was measured as described in Example 1 using the IdT2, IrT3, hairpin, and X structures illustrated in FIGS. 21 and 22. The results are shown in FIG. Both enzymes have lower RNA-dependent cleavage activity than the Tth Pol or Taq 2M enzymes. However, when the mutations cited above were introduced into these polymerases, the RNA-dependent cleavage activity was more than doubled compared to the unmodified enzyme (FIG. 25). These results demonstrate that the method according to the present invention can be used to introduce improved RNA-dependent cleavage activity into a wide range of polymerases.

3) Site-directed mutagenesis using molecular modeling and published physical studies
The crystal structure of the Taq Pol large fragment (KlentaqI) and primer / template DNA complex as determined by Li et al. (Li et al., Protein Sci., 7: 1116 [1998]) As shown in FIG. The E507Q mutation is located at the tip of the thumb subdomain, 3.8 and 18 mm away, respectively, closest to the backbone of the primer and template strands phosphate. The co-crystal structure of the Klenow fragment of DNA polymerase (Breese et al., Science 260: 352 [1993]) and Taq Pol (Eom et al., Nature 382: 278 [1996]) previously used the thumb region and the minor groove of the DNA primer / template. It was suggested that there is an interaction between Deletion of 24 amino acids corresponding to amino acids 494 to 518 of Taq Pol at the top of the thumb region of the Klenow fragment reduces DNA binding affinity more than 100-fold (Minnick et al., J. Biol. Chem., 271: 24954 [1996]). These observations are consistent with the hypothesis that the thumb region containing the E507 residue is involved in the interaction with the upstream substrate duplex.

  Co-crystal structure of Taq Pol and double-stranded DNA linked by polymerization (Li et al., Protein Sci., 7: 1116 [1998], Eom et al., Nature 382: 278 [1996]) According to the Taq Pol palm region, the W417L and G418K mutations (FIG. 17) are located approximately 25 mm from the nearest phosphate in the template and upstream chains. The same distance was observed between the similar amino acids W513 and P514 of the Klenow fragment and the template strand of DNA bound in edit mode (Breese et al., Science 260: 352 [1993]). Thus, available co-crystal experiments in this region do not suggest that there is an interaction between Taq Pol and overlapping substrates.

  Understanding the mechanism of action of the enzyme is not necessary in practicing the present invention, and the present invention is not limited to the mechanism of action, but is at positions 417 and 418 in the Taq Pol palm region. Amino acids interact with upstream substrate duplexes only when this enzyme functions as a 5 'nuclease, but no interaction with these amino acids occurs when Taq Pol is switched to polymerization mode It has been proposed. This hypothesis suggests a new form of substrate binding by the DNA polymerase referred to herein as the “5 ′ nuclease”. This hypothesis is supported by some evidence. The chimeric enzyme experiments described herein clearly separate the regions of the polymerase domain that are involved in the activity of 5 ′ nucleases and polymerases. Thus, the W417L and G418K mutations, along with the E507Q mutation, affect Taq Pol's 5 'nuclease activity on substrates with RNA target strands (Figure 15), but either RNA-dependent DNA polymerase or DNA-dependent DNA polymerase It does not affect the activity of (Fig. 16). On the other hand, a Pol I Klenow fragment of E. coli DNA that is a mutation in the active site of Taq Pol, such as R573A, R587A, E615A, R746A, N750A and D785N, which affects both polymerase activity and substrate binding affinity in polymerization mode. (Polesky et al., J. Biochem., 265: 14579 [1990], Polesky et al., J. Biol. Chem., 267: 8417 [1992], Pandey et al., Eur. J. Biochem., 214) : 59 [1993]) was shown to have little or no effect on 5 'nuclease activity. DALI program (Holm and Sander, J. Mol. Biol., 233: 123 [1993], Holm and Sander, Science, 273: 595 [1996]) and Insight II (Molecular Simulation, Naperville, IL) ), Taq Pol (Eom et al., Nature 382: 278 [1996]), E. coli Pol I and Bacillus stearothermophilus Pol I (Kiefer et al., Nature 391: 304 [1998]) polymerase domains. As a result, the palm region at amino acid positions 402 to 451 of Taq Pol, including W417 and G418, is structurally highly conserved among these three polymerases. . However, there is no structural similarity between the remaining palm subdomains. This observation suggests that this region of eubacterial DNA polymerase has an important role.

  The activities of the 5 'nuclease and the polymerase are precisely tuned and have nicks rather than gaps or overhangs that can cause deletions or insertions during Okazaki fragment processing or DNA repair if the ligase is improperly ligated to the ends. A structure must be created. According to a previously proposed model (Kaiser et al., J. Biol. Chem., 274: 21387 [1999]), when the nucleotide at the 3 ′ end of the upstream strand is sequestered by the 5 ′ nuclease domain, elongation is inhibited, Stop synthesis. Interaction with the 3 'nucleotide activates a 5' nuclease that apparently endonucleases the 5 'arm of the displaced downstream strand. This cleavage occurs by cutting exactly at the site defined by the 3 ′ nucleotide, resulting in a nick. This model requires substantial rearrangement of the substrate-enzyme complex, which rearranges the complex into the 5 'nuclease mode to separate the primer / template from the polymerase active site. Can be included.

  The interaction between the duplex formed between the template and the incoming strand and the gap formed by the finger and thumb subdomains relocates the substrate further away from the polymerase active site. It is possible to guide. Such interactions drive conformational transitions in the thumb region, bringing the template / primer duplex close to the amino acids of W417 and G418. It has been previously reported that the thumb region is significantly plastic, which can explain such changes (Breese et al., Science 260: 352 [1993], Eom et al., Nature 382: 278 [ 1996], Ollis et al., Nature 313: 762 [1995], Li et al., EMBO, J. 17: 7514 [1998]). In addition, finger domains that help open gaps, such as the transition from a “closed” structure to an “open” structure as described by Li et al. (Li et al., EMBO, J. 17: 7514 [1998]) This conformational change is consistent with this model. The 5 'nuclease binding mode was not observed in the published co-crystal structure of DNA Pol I because most of the structure was not due to the overlapping 5' nuclease substrate, but only to the polymerase domain due to the template / primer substrate. It seems that it can be solved.

For Taq Pol, Tth Pol, and TaqPol G418K / E507Q, K _m values of 200-300 nM have been determined for RNA-containing substrates. These values are higher than the K _m values assessed at <1 nM for TthPol, all due to overlapping substrates of DNA. This suggests that the RNA template adversely affects substrate binding. Low affinity can be explained by the unfavorable interaction of the enzyme with either the type A duplex adopted by the substrate with the RNA target, or the ribose 2 ′ hydroxyl group of the RNA strand. Since the 5 ′ nuclease of eubacterial DNA polymerase can be efficiently cleaved by an RNA downstream probe that is likely to have type A, the latter is more likely of these two factors (Lyamichev et al., Science 260, 778 [1993]). In addition, co-crystal studies have suggested that the template / primer duplex adopts a conformation that is close to type A by DNA polymerase in the complex (Eom et al., Nature 382: 278 [1996], Kiefer et al., Nature 391: 304 [1998], Li et al., EMBO, J. 17: 7514 [1998]). The G418K / E507Q mutation increases TaqPol's k _cat value more than twice, but has little effect on the K _m value. Such an effect is expected if the mutation causes the substrate to be placed in a more suitable direction for cleavage rather than simply to increase the binding constant.

  In addition to the mutation analysis described above, another way to study enzyme specific regions, enzyme structure-function relationships, and enzyme-substrate interactions is to examine the actual physical structure of the molecule. .

  As crystallography, NMR, and computer and software technologies advance, molecular structure research has become a viable tool for anyone interested in the organization, mechanics, and dynamics of biomolecules. Molecular modeling has deepened our understanding of the nature of interactions underlying protein structure and how proteins interact functionally with substrates. Many publications that describe various polymerases or polymerase protein sites, HIV reverse transcriptase, and other nucleic acid binding proteins have protein conformations, conformational changes, and the molecules required to function It has provided mechanistic insights into interactions.

  As an example, a report by Doubie et al. (Doubie et al., Nature 391: 251 [1998]) discloses the crystal structure of T7 DNA polymerase and which amino acid regions are likely to affect substrate binding. Provides information on which ones need to be contacted with a substrate for polymerization and which amino acids bind to cofactors such as metal ions. In this and other papers, many polymerases share not only sequence similarity but also structural homology. Stacking specific structural domains of different polymerases (eg, T7 polymerase, Klenow fragment editing complex, Taq DNA polymerase without ligand, and Taq polymerase-DNA polymerase complex) clearly identifies conserved motifs can do.

  Specifically, information from all of these structural sources and literature can be combined to create protein models that interact with DNA, RNA, or heteroduplexes. The model can then be examined to identify amino acids that may be involved in substrate recognition or contact with the substrate. Amino acids can be changed based on these observations, and screening methods such as the methods according to the invention described in the Examples are used to evaluate the effect on various activities of 5 ′ nuclease proteins.

According to the domain exchange analysis method described above, the TthDN sequence important for RNA-dependent 5 ′ nuclease activity is located in the polymerase domain of the protein. Thus, with respect to nucleic acid recognition, examining the structural data of the polymerase domain provides a method for determining the position of an amino acid, and changing the amino acid changes the RNA recognition in the 5 ′ nuclease reaction. For example, published analytical results for the primer / template to which the Pol I polymerase domain of Escherichia coli, ie, the Klenow fragment binds, were examined by analytical methods performed in the course of developing the present invention. Table 2 is the rate constant measured for the Klenow fragment and shows the effect of numerous mutations on these measurements. Amino acids that correspond or are in similar positions in TaqPol are shown in the right column. Mutations that significantly affect the interaction of Klenow fragment with DNA template or primer / template duplex also show TaqPol, and related chimeric derivatives, as shown by changes in K _d and relative DNA affinity values Alternatively, it is considered effective when created at the corresponding site of the mutant. When introduced into Klenow fragment, mutations that resulted in higher _Kd values or lower relative DNA affinity were selected and examined in TaqPol. Some of these Taq derivatives are indicated by an asterisk in the right column of Table 2, but are not limited to these.

  For Klenow mutants, such as the R682 mutant, no selection for testing was made based on DNA affinity measurements. Because molecular modeling suggested that some interaction exists between some aspect of the template / primer duplex and the amino acid. Similarly, another region of Taq polymerase (or Taq derivative) is a target for mutagenesis based on structural data and information from molecular modeling. Based on modeling, the thumb region was considered to contact the RNA template. For this reason, we searched for amino acids in the thumb region that, when modified, changed contact. For example, FIGS. 6 and 17 show that amino acids at positions 502, 504 and 507 are present at the tip of the thumb region. Altering these amino acids was thought to affect the interaction between the enzyme and the substrate. The activity screening method described in Example 1 was used to identify mutations that have beneficial effects. Several improved enzymes were created using this method. For example, the position of TaqPol H784 corresponds to the amino acid of Klenow H881, which is an amino acid in the finger region and therefore may be involved in primer / template substrate binding. Substitution of Klenow enzyme's H881 amino acid with alanine reduces the affinity for DNA to 30-40% of wild-type levels. The substitution was similarly examined with an enzyme derived from TaqPol. Starting from the Taq derivative W417L / G418K / E507Q, the amino acid at position 784 was changed from histidine (H) to alanine (A) to obtain a W417L / G418K / E507Q / H784A mutant, which was named Taq 4M. This mutant showed improved 5 ′ nuclease activity against the RNA test IrT1 (FIG. 24) test substrate (data shown in Table 3). Amino acid R587 was selected for mutagenesis because it is in the thumb region and is very close to the primer / template duplex in the computer model. When the R587A mutation was added to Taq 4M, the activity against the test IrT1 test substrate was further improved. Furthermore, if the cleavage of the X structure shown in FIG. 22 is reduced compared to the 4M mutant, this enzyme function will be further improved.

  Not all amino acid mutations that reduce DNA binding in the polymerization reaction affect 5 'nuclease activity. For example, mutations E615A, R677A, etc. that affect the amino acids present in the thumb domain and finger domain, respectively, were measured using the test substrates of FIGS. 21 and 22, and the mutants that are parents that do not have these mutations. By comparison, it has a negative or no effect on 5 'nuclease activity. The R677A mutation was added and compared to the Taq 4M mutant. The test methods described herein provide a convenient way to analyze non-invasive substrates and variants that alter the cleavage activity of invasive substrates for structures containing DNA and RNA. Thus, the present invention provides a method for identifying all suitable improved enzymes.

  Modifications that can increase the affinity of the enzyme for nucleic acid targets were also investigated. Since many of the above mutations reduced the Klenow fragment enzyme's affinity for DNA, these mutations were selected for the purpose of creating an enzyme that more easily tolerates structures containing strands other than DNA. Naturally, natural DNA polymerases have a lower affinity for RNA / DNA duplexes compared to their affinity for DNA / DNA duplexes. In the course of developing the present invention, an attempt was made to increase the general affinity of the protein of the present invention for nucleic acid substrates without restoring or increasing the preference for structures having DNA rather than RNA target strands. As a means of altering the interaction between proteins and nucleic acid substrates, the substitution of amino acids with various charges was investigated. For example, adding a positively charged amino acid residue such as lysine (K) appeared to increase the protein's affinity for negatively charged nucleic acids.

  As described above, changes in the thumb region can affect the interaction between the protein and the nucleic acid substrate. In one example, when the mutation G504K (the tip of the thumb region) was introduced into Taq 4M, the nuclease activity against the RNA target increased by 15%. The more positively charged mutations (A502K and E507K) further improved RNA target-dependent activity by 50% compared to the parent Taq4M enzyme.

  Using published research data and molecular modeling along with the results generated in the course of developing the present invention, amino acid mutations are likely to result in observable differences in one or more features of the cleavage function. The region could be identified. In this way, it is possible to target a region, but it was considered that mutations of different amino acids give different effects even in the vicinity or in the immediate vicinity of the protein. For example, the A502K substitution markedly increased the RNA-dependent cleavage activity of Taq4M, but there was little improvement when the amino acid at position 499, which was only 3 amino acids away from position 502, was changed from G to K. As can be seen from the examples, the method according to the invention mutates several amino acids in the candidate region, alone or in combination, and then uses the screening method provided in Example 1 to effect the mutation. Is to measure quickly.

  In addition to the thumb, palm, and hand regions found in the polymerase domain of these proteins, regions specific for the 5 ′ nuclease and nucleotide domains were examined. Comparative studies on various 5 'nucleases and nucleotide domains show that there are structural features common to most enzymes, even though the amino acid sequence has been dramatically altered by the enzyme. Two of these features are the helix-hairpin-helix motif (H-h-H) and the arch or loop structure. The H-h-H motif is thought to mediate non-sequence specific DNA binding. It has been found in 14 families of proteins, including nucleases, N-glycosylases, ligases, topoisomerases, and polymerases (Doherty et al., Nucl. Acid. Res., 24: 2488 [1996]). The crystallographic structure of rat DNA polymerase polβ bound to the DNA template-primer shows that nonspecific hydrogen bonding occurs between the polβ HhH motif backbone nitrogen and the hydrogen phosphate of the DNA double-stranded primer. (Pelletier et al., Science 264: 1891 [1994]). Because the H-h-H domain of the 5 ′ nuclease domain of Taq polymerase and Tth polymerase functions in the same way, mutations in the HhH region of the enzyme will not alter activity. Mutations can be introduced to change the shape and structure of the motif, or the charge on the motif can be changed to increase or decrease the affinity for the substrate.

  Another structure common to many 5 ′ nucleases obtained from a variety of derived organisms such as eukaryotes, eubacteria, archaea, and phages is the arch or loop domain. The crystal structure of the 5 'exonuclease of bacteriophage T5 showed a distinct arch formed by two helices, one positively charged and the other containing hydrophobic residues (Ceska et al., Nature 382: 90 [1995]). Interestingly, three residues, Lys83, Arg86, and Tyr82, are conserved between T5 and Taq, but they are all in the arch. These correspond to the Taq DNA polymerases Lys83, Arg86, and Tyr82. The crystal structure of Taq (5 'nuclease) has also been determined (Kim et al., Nature 376: 612 [1995]).

  The crystal structure of flap endonuclease-1 derived from Methanococcus jannasi also exhibits such a loop motif (Hwang et al., Nat. Struct. Biol., 5: 707 [1998]). The crystal structure of the Mja FEN-1 molecule backbone can be overlaid with T5 exonuclease, Taq 5 'exonuclease, and T4 RNaseH. An interesting feature common to all of these is the long loop. The FEN-1 loop consists of a number of positively charged and aromatic residues, forming a hole large enough to accommodate a single-stranded DNA molecule. The corresponding region in T5 exonuclease is the three helices that form a helical arch. In T5 exonuclease, the size of the hole formed by the helical arch is smaller than half of the hole formed by the L1 loop of Mj FEN-1. In T4 RNaseH, or Taq 5 'exonuclease, this region is disordered. Some arch regions bind to the metal, while other arch regions contact the nucleic acid substrate. A sequence comparison of the amino acid sequences of six 5 'nuclease domains from the pol family of DNA polymerases reveals that there are six highly conserved sequence motifs containing ten acidic residues (Kim et al. , Nature 376: 612 [1995]).

  The effect of modification in the arch area was investigated. In Taq polymerase, the arch region is formed by amino acid positions 80-95 and 96-109. Site-directed mutagenesis was performed in this arch region. A sequence comparison of the amino acid sequences of the FEN and polymerase 5 'nucleases suggested that three amino acid substitution mutations, P88E, P90E and G80E, were designed. These substitutions were made above with the Taq4M polymerase mutant as the parent enzyme. As a result, the background activity against the HP and X substrates shown in FIG. 22 was very strongly suppressed in all mutants, but also the desired 5 ′ nuclease activity against the correct substrates (IdT and IrT, FIG. 24). descend. Despite the sequence homology between Taq polymerase and Tth polymerase, they show completely different activities against HP and X substrates. In addition, comparing the sequence of the arch region of Taq polymerase and Tth polymerase reveals slight differences from a wide range of the same region. These differences led to the design of mutations L109F and A110T, Taq4M was used to create Taq4M L109F / A110T, and mutations Taq4M L109F / A110T / A502K / G504K / E507K / T514S were used to create Taq4M. These two mutations drastically changed the Taq4M enzyme, making it more similar to the Tth enzyme in terms of background substrate specificity, but with little change in 5 'nuclease activity on DNA and RNA substrates. .

4) Directed random mutagenesis As mentioned above, physical studies and molecular modeling can be used alone or in combination to make visible differences in one or more features of the cleavage function by amino acid mutations. A region of a protein that appears to be highly potent can be identified. The previous section described how to use this information to select mutation-specific amino acids and combinations of amino acids. Another way to create an enzyme with an altered function is to introduce mutations randomly. Such mutations can be introduced by a number of methods known in the art, PCR amplification (REF) performed under conditions such that the preferred nucleotide is misincorporated, primers with degenerate regions Amplification methods using (ie, different at each base position, but in the same reaction, otherwise the same oligonucleotide has a different base), and chemical synthesis, but are not limited to these. A number of random mutagenesis methods are known in the art (Del Rio et al., Biotechniques, 17: 1132 [1994]) and can be incorporated into the production of enzymes according to the present invention. The discussion of specific methods of mutagenesis included herein is merely provided as an example and is not limiting. When random mutagenesis is performed so as to mutate only a specific region of the entire protein, it can be expressed as “directed random mutagenesis”. As described in the experimental examples, directed random mutagenesis was applied to mutate the HhH and thumb domains of the previously generated enzyme mutants. These domains were chosen to provide examples of the method and are not intended to limit directed random mutagenesis to a particular domain or a single domain. The method can be applied to any domain or whole protein. The thus modified protein was tested for cleaving activity in the screening reaction described in Example 1 using the test substrate illustrated in FIG. 22 and FIG. The results are shown in Table 6 and Table 7.

  Random mutagenesis was performed on the HhH region using the parent TaqSS or TthDN H785A mutant. None of the 8 mutants produced showed any improvement compared to the parent enzyme (Table 6). In fact, mutating the region between residues 198-205 shows about 2-5 times lower activity on DNA and RNA substrates, indicating that this region is essential for substrate recognition. Suggests. Mutagenesis in the thumb region resulted in new mutations that improved 5 'nuclease activity by 20-100% for DNA targets and about 10% for RNA targets (Table 7).

  In each of the different subdomains, a number of amino acids play a role in contact with the substrate. These mutagenesis can alter substrate specificity by altering substrate binding. Furthermore, mutations introduced into nucleic acids that are not in direct contact with the substrate can also change the substrate specificity over a longer range or by a general conformational modification effect. These mutations are introduced by any method known in the art, including but not limited to random mutagenesis, site-directed mutagenesis, and chimeric protein production. Can do.

  As noted above, many methods for random mutagenesis are known in the art. Methods applicable to the directed random mutagenesis described herein can be applied to all genes. Still other useful chimeric constructs are available from molecular breeding methods (eg, US Pat. No. 5,837,458, and WO 00/18906, WO 99/65927, WO 98/31837, and See WO 98/27230, which can be made using (incorporated herein by reference in their entirety). Regardless of the mutagenesis method chosen, the rapid screening methods described herein provide a quick and effective method of identifying beneficial mutations in a large population of recombinant molecules. This makes random mutagenesis a tractable and practical tool for creating large populations of modified 5 'nucleases with beneficial improvements. The cloning and mutagenesis methods used for the enzymes used as examples can be applied to other thermostable and non-thermostable type A polymerases. This is because the similarity of DNA sequences is very high between these enzymes. One skilled in the art will appreciate that sequence differences may require changing the cloning method, for example, it may be necessary to use different restriction endonucleases to create a chimera. Selecting existing selective sites or introducing selective sites by mutagenesis is a well-established method and is known to those skilled in the art.

  Enzyme expression and purification can be performed by various molecular biological methods. The following example teaches one such method. However, it should be understood that the present invention is not limited by the methods of cloning, protein expression, or purification. The present invention envisions that the nucleic acid construct can be expressed in a suitable host cell. Numerous methods of genes can be utilized to link various promoters and 3 ′ sequences to the gene construct to achieve efficient expression. .

5) Site-directed mutagenesis In some embodiments of the invention, heterologous domains can be used using any suitable technique (eg, but not limited to, one or more of the techniques described above). An improved cleavage enzyme (SEQ ID NO: 221) with Thus, in some embodiments, site-directed mutagenesis methods (eg, primer-specific mutagenesis methods using commercially available kits such as Transformer Site Directed mutagenesis kits (Clonetech)) In use, multiple mutations can be created throughout the nucleic acid sequence to generate a nucleic acid encoding a cleavage enzyme according to the present invention. In some embodiments, multiple primer-specific mutagenesis steps can be performed in parallel to create a nucleic acid encoding a cleavage enzyme according to the present invention.

  In some embodiments, a plurality of primer-specific mutagenesis steps are performed on selected sites of the nucleic acid sequence to create mutations at selected sites of the cleavage enzyme according to the present invention. In another embodiment, a nucleic acid having a mutation at a selected site is recombined with a nucleic acid having a mutation at another selected site (eg, cloning, molecular breeding, or other set known in the art). Thereby producing a nucleic acid encoding a cleavage enzyme according to the present invention with mutations at a plurality of selected sites. Any of the above methods, including, but not limited to, site-directed mutagenesis and random mutagenesis, or a combination of the methods, Mutations in each can be introduced.

  For example, in one exemplary embodiment of the invention, multiple primer-specific mutagenesis is performed on the nucleic acid sequence of SEQ ID NO: 104 (see Example 7 for construction of SEQ ID NO: 104). Thus, the nucleic acid sequence of SEQ ID NO: 222 (nucleic acid sequence encoding the cleavage enzyme of SEQ ID NO: 221) is prepared. In some embodiments, each mutation is introduced using a separate mutagenesis reaction. The reaction is continuously performed so that the obtained nucleic acid (SEQ ID NO: 222) contains all the mutations. In another exemplary embodiment of the invention, as described above, by performing multiple primer-specific mutagenesis on the nuclease site of SEQ ID NO: 111 (eg, illustrated in FIG. 6), SEQ ID NO: 222 A nucleic acid sequence of Next, using the recombination method described in Example 4, the mutant nuclease site was linked to the “polymerase” site of SEQ ID NO: 104 at the NotI site, having SEQ ID NO: 222, and cleavage of SEQ ID NO: 221 Create a single nucleic acid encoding the enzyme. After mutagenesis, the resulting modified polypeptide is produced and cleaved using a suitable assay (eg, but not limited to those described in Examples 1 and 6). Check for activity. In some embodiments, the nucleic acid sequence encoding the cleavage enzyme of SEQ ID NO: 221 (eg, SEQ ID NO: 222) is further modified using appropriate methods.

V. Design of detection reaction of RNA target by INVADER assay
Methods related to the design of the INVADER assay for RNA target detection will vary depending on the specific assay requirements. For example, in certain embodiments, it is preferred that the RNA to be detected or analyzed be present at a low level in the test sample and thus be highly sensitive (ie, have a low detection limit or LOD). In other embodiments, the RNA is abundant and the sensitivity of the detection assay need not be particularly high. In certain embodiments, it is preferred that the RNA to be detected is similar to other RNA that is not intended for detection in the sample and the assay is highly selective. In other embodiments, it is preferred that multiple similar RNAs are detected in a single reaction, thus providing an assay that does not exhibit selectivity with respect to differences between these similar RNAs.

  In certain embodiments, it is particularly preferred not to detect DNA molecules associated with the target RNA molecule in the reaction. In some embodiments, this is accomplished by designing a probe set for an INVADER assay against an RNA splice site such that only properly spliced mRNA provides a selected detection target site. In other embodiments, the sample is manipulated such that the DNA remains double stranded (eg, does not heat the nucleic acid or leave it in denaturing conditions) and therefore does not serve as a target in an INVADER assay reaction. In other embodiments, nuclei lysing cells under intact conditions cause cytosolic mRNA to be released into the lysate for detection by the assay, but prevent and prevent detection of genomic DNA.

In some embodiments, the INVADER assay is utilized for the detection or quantification of total RNA containing a specific mutation in the sequence (eg, mutation, SNP, specific splice site). In such an embodiment, the base or sequence position to be detected is a determinant in the selection of the site for the hybridized INVADER assay probe set. In other embodiments, any portion of the RNA target can be used to indicate the presence or amount of total RNA (eg, as in gene expression analysis). In this case, the probe set has been found to target a portion of the RNA selected for maximum performance as a target in the INVADER assay (eg, particularly accessible for probe hybridization). Site). The discussion of INVADER assay probe design is divided into the following sections:
i. Target site selection based on accessibility
ii. Target site selection based on selectivity
iii. Oligonucleotide design
a. Target specific region: length and melting temperature
b. Non-complementary region
c. Folding and dimer analysis
iv. Evaluation of assay performance
v. Design and assay optimization

i. Target site selection based on accessibility Consideration of site selection for detection is the availability of the target site in the hybridization of the probe set of the assay. Simply using complementary oligonucleotides randomly selected for a given RNA target without prior knowledge of the region of RNA that allows efficient hybridization can be an ineffective approach . For example, it has been estimated that 18-20 species tested with target RNAs containing antisense oligonucleotides based on random design yield one oligonucleotide that significantly inhibits gene expression (Sczakiel, Fronteirs in Biosciences 5: 194 [2000]; Patzel et al., Nucleic Acids Res., 27: 4328 [1999]; Peyman et al., Biol. Chem. Hoppe-Seyler 367: 195 [1995]; Monia et al., Nature Med., 2: 668 [ 1996]). The secondary and tertiary structure of RNA is believed to be a major cause affecting the ability of oligonucleotides to bind to target regions of RNA (Vickers et al., Nucleic Acids Res., 28: 1340 [2000]; Lima et al., Biochemistry 31: 12055 [1992]; Uhlenbeck, J. Mol. Biol., 65:25 [1972]; Freier and Tinoco, Biochemistry 14: 3310 [1975]). This is due to hybridization kinetics and thermodynamics that cleave any structural motif of RNA and instead hybridize to complementary DNA oligonucleotides (Patzel et al., Nucleic Acids Res., 27: 4328 [1999]. ]; Mathews et al., RNA 5: 1458 [1999]). Thus, the ability to identify regions of RNA that are “accessible” for hybridization is important for effective oligonucleotide design and selection.

  There are multiple experimental and theoretical methods available for identifying accessible regions in RNA. These include RNaseH footprinting (Ho et al., Nature Biotechnology 16:59 [1998]; Mateeva et al., Nucleic Acids Res., 25: 5010 [1997]; Mateeva et al., Nature Biotechnology 16: 1374 [1998]), oligonucleotides Complementary arrays of libraries (Southern et al., Nucleic Acids Res., 22: 1368 [1994]; Mir and Southern, Nature Biotechnology 17: 788 [1999]), ribozyme libraries containing internal guide sequences with random hexamers ( Campbell and Cech, RNA 1: 598 [1995]; Lan et al., Science 280: 1593 [1998]), and RNA and DNA structure prediction computer program (Sczaldel, Frontiers in Biosciences 5: 194 [2000]; Patzel et al., Nucleic Acids Res., 27: 4328 [1999]; Zuker, Science 244: 48 [1989]; Walton et al., Biotechnol. Bioeng., 65: 1 [1999]). Recently, for hybridization, new methods have been developed that utilize primer extension to identify accessible sites in RNA. A target nucleic acid (eg, mRNA target nucleic acid) is contacted with a plurality of primers containing the 3 ′ region of the degenerate sequence, and a primer extension reaction is performed. If the target nucleic acid is an RNA molecule, the preferred enzyme utilized for the extension reaction is a reverse transcriptase that produces a DNA copy of the RNA template. The presence or absence of a folded structure in the target nucleic acid affects the initiation and / or efficiency of the extension reaction. The extension product of the primer is analyzed to create a map of accessible sites. For example, if a primer is complementary to a sequence related to a folded structure, a specific extension product does not occur. The region of the target nucleic acid where the primer cannot hybridize and no extension product is produced is considered an inaccessible site. In contrast, the extension product indicates that the primer was able to bind to an accessible region of the target nucleic acid. In the present specification, such a method is referred to as “reverse transcription by a random oligonucleotide library” or “RT-ROL” (HT Allawi et al., RNA 7 (2): 314-27 [2001]). . The use of physical measurements such as RT-ROL or array hybridization provides the most direct evidence of accessibility at sites on the RNA strand. In general, the INVADER assay for accessible regions produces a stronger signal for a given amount of RNA than the assay for less accessible regions of the RNA strand. For the detection of rare RNAs (eg, less than about 5,000 to 10,000 copies per INVADER assay reaction), or in assays where the best possible (ie lowest) detection limit is desired, RT-ROL or another physical It is beneficial to begin assay design by analyzing RNA structure using genetic analysis.

  In other embodiments, it is particularly important that the assay design is simpler than building a low LOD assay. Using structure prediction software, you can easily determine which part of RNA is single-stranded and more accessible for probe hybridization. First, enter the RNA sequence to be detected into an electronic file. This can be entered manually or imported from a file (eg, a sequence data file or a word processor file). In some embodiments, the sequence is downloaded from a database such as GenBank or EMBL. Next, mfold (Zucker, Science 244: 48 [1989]), OligoWalk (Mathews et al., RNA 5: 1458 [1999]) and variants of both (Sczakiel, Frontiers in Biosciences 5: 194 [2000]; Patzel et al., Nucleic Acids Res., 27: 4328 [1999]; Walton et al., Biotechnol Bioeng., 65: 1 [1999]) and other programs can be used to analyze RNA sequences.

Mfold analysis for prediction of target RNA structure
Multiple methods using the output of mfold analysis can help identify accessible regions of RNA target molecules. In one embodiment, an “SS count” file is created using the mfold program to identify the regions that are least involved in internal chain base pairing. In another embodiment, the “.ct” file is created using the mfold program. This file is used as input information in RNAStructure 3.5 when performing OligoWalk analysis. In a preferred embodiment, the sequence to be detected is input to mfold for the purpose of use in either. In a preferred embodiment, settings used for mfold analysis include the following.
・ Folding temperature: 37 ℃ (even if INVADER reaction is not the same temperature)
・ Semi-optimal%: 5
・ Folding number: 50
・ Window parameter: Default ・ Maximum distance between paired bases: Unlimited ・ Select batch folding ・ Enter e-mail address to send the result when preparation is completed ・ Resolution: High ・ Structure format: Base / base frequency: default / rotation angle of structure: 0
・ Comments on structure: SS count ・ 1M NaCl (Australian mfold Internet site only)

  When the results are ready, send an e-mail message containing a web address about the results. The only file required for subsequent INVADER assay design analysis is the SS count file, which is then downloaded from the page containing the results. An example of mfold analysis using the human ubiquitin GenBank entry code (# 4506712) is shown below.

SS count analysis to identify accessible sites Next, import the SS count file into an Excel spreadsheet file and select from the following options: Data type = Delimiter <Press Next>; Delimiter = Select (x) Space; (x) Process multiple delimiters with a single <Press Next>. Data format column = general. By selecting these options, they are imported into three Excel data columns (Figure 39). The first row of the first column is the total number of stable structures that mfold finds under the parameter. In the example of ubiquitin, 12 different structures were found.

  The remainder of the first column is the positions of RNA nucleotides numbered from 1. The second column is the raw SS count and represents the number of unpaired bases where the corresponding base is present as part of the secondary structure. The third column is the analyzed RNA sequence and identifies the numbered base at each position. By examining the bases associated with fewer structures (ie, the bases shown in column 3 corresponding to the higher number in column 2), the internal chain is more likely to be an unpaired base, and therefore the INVADER assay Can be used to identify the region of mRNA (identified by the position number in column 1) that is likely to be detected.

  One way to look at the data is to calculate the running average SS count for a 10-nucleotide strand of RNA (Ave (10) index) and Ave (10) for the base pair position (see, eg, Figure 48) Chart the index.

  As another plot, the nucleotide positions are graphed against the Ave (10) index expressed as a percentage of the total number of structures found by mfold (Figure 39). In the raw SS count table, the RNA region with the higher Ave (10) index correlates with the lower number of predicted folded structures. By charting or graphing the Ave (10) index, you can reduce the complexity of the data, and find the more probable RNA parts that are longer unstructured. For example, the user can retrieve all the main peak areas in the region suitable for INVADER assay probe design from the running average graph. In this way, a candidate list for SS count is created. In the case of human ubiquitin, one main peak is found, and about 6 other peaks are found (FIG. 39).

  In the next step, we return to the raw (ie not running average) SS count data within each peak area and refer to it to identify residues whose running average value changes. This is a local “turn” and is generally a good candidate residue located at the cleavage site of the assay probe set. For example, in human ubiquitin, residue G119 is found in a small local turn in a globally accessible region (Figure 40). The INVADER assay probe set with a cleavage site at this position shows good performance in detecting this RNA. Although G114 has a higher% Ave (10) value, setting the cleavage site to G114 did not result in better detection. Without limiting the present invention to a particular mechanism, and understanding the mechanism for carrying out the present invention is unnecessary, there is more accessibility to the probe or INVADER oligonucleotide in this region compared to the position of G119. Low is likely the cause.

OligoWalk structure prediction using RNAStructure 3.5 for identification of accessible sites In one embodiment, oligonucleotide OligoWalk is a program that is a module of the software “RNAStructure” (Mathews et al., RNA 5: 1458 [1999]). Select sites that are more accessible for binding. OligoWalk uses a set of thermodynamic parameters for RNA and DNA and their hybrids in an mfold-based algorithm for RNA secondary structure prediction (Zucker, Science 244: 48 [1989]) (Allawi and SantaLucia, Biochemistry 36: 10581 [1997]; Mathews et al., J. Mol. Biol, 288: 911 [1999]; Sugimoto et al., Biochemistry 34: 11211 [1995]). OligoWalk estimates the overall thermodynamics of antisense oligomers to RNA by taking into account the thermodynamics of cleaving any structural motif in the RNA target or antisense oligonucleotide, and the antisense oligonucleotide In this design, the most suitable RNA target region is predicted. The affinity of the oligomer for the target is expressed as the total Gibbs free energy change of the target binding to the self-structured oligomer and the oligomer-target complex. This free energy is usually a negative number, represents a suitable bond, and is expressed in units of “kcal / mol”. OligoWalk analysis is performed using an oligonucleotide size of 8-15 bases similar to the average length of the analyte-specific region of the signal probe. Plot total binding energy against RNA length to obtain the peaks and troughs of the graph. A negative minimum generally indicates the most preferred site for oligonucleotide binding. The most inaccessible areas have positive binding energy values and are generally unfavorable sites for designing assay probes.

In a preferred embodiment, the RNA energy 3.5 OligoWalk module is used to perform an 8-base OligoWalk using the following settings to determine the binding energy.
-Destructive local structure-Includes quasi-optimal structure-Oligo length: 8 nucleotides-Oligo concentration: 100 nM
・ Oligotype: DNA
・ Walk all target RNAs

  Once these parameters are set, select and open the sequence file to be folded (mfold ".ct" output file). Once the array is folded, a report can be generated using the output menu. Import the report into Excel and plot the data generated above. In a preferred embodiment, OligoWalk data is graphed with SS count data. Regions that exhibit a free energy minimum (ie, a negative maximum number) are usually likely to be accessible for hybridization. In a preferred embodiment, the 3 'end of the probe oligonucleotide and the majority of the target binding region are complementary to the accessible region of the target RNA. In particularly preferred embodiments, the majority of the binding sites for the corresponding INVADER oligonucleotide are in the same accessible region. In another preferred embodiment, the binding site for the INVADER oligonucleotide is in a nearby accessible region.

Usually, it can be set at a position where the INVADER oligonucleotide binds to a site with lower accessibility. Without limiting the present invention to a particular mechanism, it is generally known that INVADER oligonucleotides are longer than probe oligonucleotides used in INVADER assay reactions. This is because it is usually designed to remain bound to the target at the reaction temperature and is selected to have a T _{m of} about 12-15 ° C. higher than the corresponding probe. Therefore, INVADER oligonucleotides are less dependent on the accessibility of target binding sites because they more easily cleave local target structures.

  When designing an oligonucleotide for an INVADER assay, when selecting from accessible sites, the base composition of the site is also considered. Any of the binding sites for the assay oligonucleotide that have more than 4 or 5 nucleotide repeats in a row (eg ... AAAA ... or ... CCCC ...) It is known to reduce the performance of a probe set in an assay (eg, by increasing background or reducing specificity). Thus, in a preferred embodiment, ones that normally contain 4 bases or more repeats are avoided. Also consider the effect of base composition on the length of the oligonucleotide in the probe set. In many cases, when targeting an AT-rich sequence, when reacting at a given temperature, compare the length of the target oligonucleotide to a sequence with a more uniform distribution of AT and GC bases. Thus, it is necessary to use longer oligonucleotides. Longer oligonucleotides tend to form internal chain and dimeric structures. Therefore, if a detection region is possible, it is preferred that the distribution of A-T bases and G-C bases in the target region is approximately the same (ie, a G-C content of about 50%). In a particularly preferred embodiment, the position distribution of A-T and G-C is uniformly distributed throughout the binding site (eg, no half is A-T and the other half is G-C).

ii. Selection of Target Sites Based on Selectivity In some embodiments, probe sets are designed to examine highly homologous or closely related RNA targets (ie, targets that are very similar in sequence). In such an embodiment, RNA or homologous cDNA sequences are compared using a parallelization program such as MEGALIGN (DNAstar Madison, WI).

  In some embodiments, selectivity is obtained by designing a probe set that detects splice sites. Splice sites can be identified by parallelizing cDNA and gene sequences using a parallelization program (eg, MEGALIGN) using the BLAST menu on the NCBI website (BLAST2 sequence). Also, splice sites are often listed in GenBank reports (intron / exon sites). The INVADER assay oligonucleotide set is designed so that the probe and INVADER oligonucleotide are complementary to the coding strand (mRNA) and the cleavage site is usually as close to the splice site as possible. In some embodiments, the accessibility of different splice sites within the mRNA is analyzed as described above. In a preferred embodiment, a probe set is designed to detect one or more splice sites that are more accessible compared to the accessibility of other splice sites within the same RNA target.

  In a design embodiment that eliminates the detection of RNA associated with the target RNA, the sequence is examined in order to identify unique bases in the target RNA relative to other similar sequences that distinguish the target. Usually, the unique base is positioned to hybridize to the 5 ′ end of the target specific region of the probe oligonucleotide. In some embodiments, two adjacent bases are unique to the target compared to the associated RNA. If two unique bases are present adjacent to a suitably accessible portion of the target RNA, it is preferable to utilize these bases in the surrounding sites for designing probes and INVADER oligonucleotide sets. In some embodiments, these two unique bases are positioned so that there is a probe cleavage site between the two base pairs formed with the probe. In other embodiments, one of the unique bases is present at the end of the hybridization site of the INVADER oligonucleotide (ie, the position that base pairs with the penultimate residue at the 3 ′ end of the INVADER oligonucleotide). ).

  In some embodiments, the assay is designed to include detection of RNA that is similar but not identical to the target RNA. If the assay is designed for comprehensive detection, the sequences to be compared are examined to identify sites with complete homology. Such a design can be performed for the purpose of detecting a sequence homologous between the same species or between species. Usually, the most homologous region is selected as a hybridization target for the probe oligonucleotide. Usually, some variation can be tolerated. For example, this is the case if it is not a base that hybridizes to the 5 'end of the target specific region of the probe. In some embodiments, the use of a degenerate base in an oligonucleotide of an INVADER assay results in a mutation (e.g., using a mixture of bases at positions within a synthetic probe, INVADER and / or a stacker oligonucleotide, the mixture being , Select to complement a mixture of specific bases present in the set of related target RNAs).

スタッカーを利用する他の態様においては、安定性のより低いスタッキング相互作用が好ましい。このような場合には、プローブの3'塩基および／もしくはスタッカーの5'塩基を、スタッキング相互作用がより不安定になるように選択する。ある態様においては、プローブの3'塩基および／もしくはスタッカーの5'塩基を、標的鎖に関してミスマッチを有し、スタッキング相互作用の強度を低下させるように選択する。 iii. Oligonucleotide design
a. Target Specific Region: Length and Melting Temperature As described in Section I (a) above for oligonucleotide design, in some embodiments, the length of the analyte specific region depends on the temperature at which the reaction is performed. Calculated optimal reaction temperature ( _Tm plus enzyme and other reaction condition effects) starting from the desired position (eg, mutation position or splice site in target RNA, or site with low free energy value in OligoWalk analysis) An iterative method is used in which the length of the ASR is increased by one base pair until the salt correction to compensate for matches the desired reaction temperature. Usually, when the stacking oligonucleotide is not used, the calculated T _m is about 60 ° C., and when the stacking oligonucleotide is used (the stacking oligonucleotide is usually the T of the adjacent probe oligonucleotide). _m is increased by about 5-15 ° C.), and the probe is selected to be an ASR with T _m of about 50-55 ° C. If the position of the mutation or splice site is the starting point, add it to the 3 'end of the probe. Alternatively, if the 3 ′ end of the probe is located at the most accessible site, it is added in the 5 ′ direction. In the embodiment using the stacker oligonucleotide, it is preferable to design the probe so as to include a 3 ′ base having an interface having a stable stacking interaction with the 5 ′ base of the stacker oligonucleotide. The stability of coaxial stacking is highly dependent on the identity of the stacking base. Overall, the qualitative trend of coaxial stacking decreases according to the following order:

In other embodiments utilizing a stacker, less stable stacking interactions are preferred. In such cases, the 3 ′ base of the probe and / or the 5 ′ base of the stacker is selected so that the stacking interaction is more unstable. In some embodiments, the 3 ′ base of the probe and / or the 5 ′ base of the stacker is selected to have a mismatch with respect to the target strand and reduce the strength of the stacking interaction.

The same principle is used for the design of INVADER oligonucleotides. Briefly, starting from position N, then starting from residue N-1 until the stability of the INVADER-target hybrid exceeds that of the probe (and thus the planned assay reaction temperature) Additional residues complementary to the RNA are added upstream. In a preferred embodiment, the stability of the INVADER-target hybrid is 12-15 ° C. above that of the probe. Usually, the INVADER oligonucleotide is selected so that the T _m is close to 75 ° C. Such calculations can be supported using software such as INVADERCREATOR (Third Wave Technologies, Madison, Wis.) Or Oligonucleotide 5.0.

Similar design principles are utilized when using stacking oligonucleotides. Usually, the stacking oligonucleotide is designed such that the stacker / target helix that hybridizes at the site adjacent to the 3 ′ end of the probe oligonucleotide and the formed stacker / target helix can be coaxially stacked with the probe / target helix. The sequence is selected so that the T _m calculated in the calculation using a natural base is about 60 to 65 ° C. However, stacking oligonucleotides are usually synthesized using only 2′-O-methyl nucleotides, and therefore for an actual T _m close to 75 ° C., the actual T _m is about 0.8 per base than calculated. ℃ higher.

  In some embodiments, the second reaction includes an ARRESTOR oligonucleotide. In order to eliminate the interaction between the first probe and the second target strand, the ARRESTOR oligonucleotide is utilized to sequester the remaining uncleaved probe from the first reaction in the second reaction. ARRESTOR oligonucleotides are usually 2'-O-methylated, are essentially complementary to all target-specific regions of individual probes, and probe flap regions (eg, from +1 base to arm 6 nucleotides counted toward the 5 ′ end of the first portion).

b. Non-complementary region Selection of the 5 'arm of the probe If a non-complementary arm of the probe is present, this is preferably selected (in the repetitive step according to the above method) and the second reaction is carried out at the specific reaction temperature. can do. In the second reaction, the second probe is usually used repeatedly and it is necessary to stably bind the cleaved 5 ′ arm (acting as an INVADER oligonucleotide) to the second target strand.

Selection of mismatches at the 3 ′ end of the INVADER oligonucleotide In a preferred embodiment, the 3 ′ base of the INVADER oligonucleotide is not complementary to the target strand and is listed in the following order of preference (INVADER oligonucleotide 3 ′ base / target base: Select).
C in target: C / C> A / C> T / C> G / C
A in target: A / A> C / A> G / A> T / A
G in target: A / G> G / G> T / G> C / G
U in target: C / U> A / U> T / U> G / U

c. Folding and dimer analysis In one embodiment, the oligonucleotides utilized in the INVADER assay are considered for possible intermolecular and internal molecular formation in the absence of the target RNA. This is usually preferred for probe assays with less predicted intermolecular and internal molecular interactions. In one embodiment, the program OLIGO (eg, OLIGO 5.0, Molecular Biology Insights, Inc., Cascade, CO) is utilized for such analysis. In another embodiment, the program mfold is used for the analysis. In yet another embodiment, the RNAStructure program can be used for dimer analysis. In the next section, the use of these programs for analysis of oligonucleotides in the INVADER assay will be described in turn.

OLIGO 5.0 analysis of probe structure and interaction prediction
Analysis of INVADER oligonucleotide using OLIGO 5.0 includes the following steps. All menu choices are shown in capital letters.

1. Start OLIGO 5.0 and open a sequence file for each mRNA to be analyzed. This is done using a menu consisting of the following choices:
・ Select FILE-> NEW ・ Paste the longest available sequence ・ Select ACCEPT & QUIT (F6)

2. Make program settings default
Select FILE->RESET-> ORIGINAL DEFAULTS

3. Identify the probe or oligonucleotide. • Select OLIGO LENGTH of about 16 nucleotides (use the ctrl-L key to open this option menu).
• Move the cursor indicating the 5 'end of the Current Oligo until the 3' end is at the candidate residue at the cleavage site.
• Select ANALYSE-> DUPLEX FORMATION-> CURRENT OLIGO (ctrl-D) to roughly determine the extent of dimer and hairpin formation.
Confirm that the length of the test area corresponds to the desired reaction temperature (eg, use the calculation of T _m by the method described in I (c) of the detailed description of the invention, optimization of reaction conditions) by).
Select the OLIGO 5.0 “LOWER” button to copy the antisense sequence (this is the analyte-specific region of the actual probe oligonucleotide and is antisense to the RNA strand).
• Import into a database file.
・ Save to computer memory.

4. Identify the INVADER oligonucleotide • Select the sequence adjacent to the probe oligonucleotide identified in step 3.
• For OLIGO LENGTH, select approximately 24 nucleotides.
Confirm that the length of the test area corresponds to the desired reaction temperature (eg, using the calculation of T _m by the method described in I (c) of the detailed description of the invention, optimization of reaction conditions) (About 75 ° C. for INVADER oligonucleotides). Select the OLIGO 5.0 “LOWER” button to copy the corresponding antisense sequence (which is the analyte-specific region of the actual INVADER oligonucleotide).
• Import into a database file.
・ Save to computer memory.

5. Export the cleaved arm sequence and the INVADER oligonucleotide mismatch sequence / probe / oligonucleotide as upstream primer.
Export the INVADER oligonucleotide as a downstream primer.
• Run EDIT UPPER PRIMER to add candidate arm sequences (eg, selected as described above).
Confirm that the arm sequence does not form a new secondary structure (analyzed as described above).
Run EDIT LOWER PRIMER to add 3 'mismatched nucleotides that overlap the cleavage site (selected according to the guidelines for this mismatched base shown above).
In the “Print / Save Options” box, select all of the upstream and downstream primers.
• Confirm upstream (probe) and downstream (INVADER) oligonucleotide PRINT ANALYSIS and lack of stable secondary structure.
Save both mRNA and oligonucleotide sequence database files before stopping the program.

  In general, oligonucleotides that have been detected to have intramolecular formation that are less stable than -6ΔG are preferred. Since less stable structures are less likely to be substrates for the CLEAVASE enzyme, cleavage of such structures is unlikely to contribute to the background signal. Probes and INVADER oligonucleotides with low affinity for each other are available for binding to the target and give the highest cycling efficiency.

The T _m of a dimerization probe (ie, a probe molecule that hybridizes to another probe molecule) is ideally lower than the T _{m of} a probe that hybridizes to the target and is usually high for performing INVADER assay reactions At temperature, the probe can selectively hybridize to the target sequence. Similarly, the T _m of a hybridized INVADER oligonucleotide itself or probe molecules, should be lower than the T _m of INVADER oligonucleotide / target. The dimer's T _m (ie, probe / probe and probe / INVADER oligonucleotide) is preferably 25 ° C. or lower, preferably reducing the probability of formation at the intended reaction temperature.

  The melting temperature for each of these complexes can be determined using the method described in I (c) Optimization of reaction conditions in the detailed description of the invention above, or OLIGO software. After selecting multiple RNA site and INVADER assay oligonucleotide set candidates according to the steps outlined above, a degree according to the preferred selection rule, eg, mean value plot of SS counts (peak, valley, otherwise) Ranking of candidate oligonucleotide sets can be performed according to the position in and the energy prediction of the probe and INVADER oligonucleotide interactions. In certain embodiments, the ranked probe sets are tested in rank order to identify one or more sets that have suitable performance in an RNA INVADER assay. In other embodiments, multiple higher rank sets (eg, 2, 3 or more) are selected and tested to quickly identify one or more sets with suitable or desired performance. .

Probe structure Mfold analysis and interaction prediction Probe analysis and INVADER oligonucleotide interaction can be performed using DNA mfold by Michael Zuker. mfold is available from Rensselaer Polytechnic Institute bioinfo.math.rpi.edu/˜mfold/dna/forml.cgi. Do not change the default ion conditions, select a temperature of 37 ° C, set the% quasi-optimal setting to 75, and perform the analysis. Each program (eg, probe, INVADER oligonucleotide, stacker, etc.) is folded using a program to confirm the formation of a single molecular structure (eg, hairpin). The energy related to the monomolecular structure calculated by mfold can be used as it is without further calculation.

、さらに続けて、同一のオリゴヌクレオチド配列、再び5'から3'を入力することによって、与えられたオリゴヌクレオチドの2分子構造形成を評価する。これらのTが一本鎖のままであり、このスペーサーにおけるCとGの並びが塩基対を形成するために必要な束縛値を入力する。強制的に塩基対を形成させるためには、コマンド「F」を利用し、塩基対の形成を禁止するためにはコマンド「P」を用いる。塩基対の強制もしくは禁止の位置は5'末端からカウントする。例えば、目的の配列が20量体であれば、以下を入力する。
F 21 0 5 ｛これによって、C21からC25までのCが塩基対を作る｝
P 26 0 4 ｛これによって、T26からT29までのTが一本鎖を作る｝
F 30 0 5 ｛これによって、G30からG34までのGが塩基対を作る｝ Oligonucleotide sequence (5 'to 3') followed by a sequence that forms a small, stable hairpin

Further, the bimolecular structure formation of a given oligonucleotide is evaluated by entering the same oligonucleotide sequence, again 5 ′ to 3 ′. These Ts remain single-stranded, and the sequence of C and G in this spacer inputs the binding value necessary to form a base pair. The command “F” is used to forcibly form a base pair, and the command “P” is used to prohibit the formation of a base pair. Base pair forced or prohibited positions are counted from the 5 'end. For example, if the target sequence is a 20mer, enter:
F 21 0 5 {by which C from C21 to C25 make a base pair}
P 26 0 4 {Thus, T from T26 to T29 forms a single strand}
F 30 0 5 {Thus, G from G30 to G34 makes a base pair}

によって形成したコア・ヘアピンは、以下の熱力学量を有する：
ΔG＝-5.3；ΔH＝-37.8；ΔS＝-104.8。 Examining the resulting structure, the total (ie possible structural thermodynamic value = mfold structural thermodynamic value-core hairpin thermodynamic value) to the stability of the central spacer hairpin (ie thermodynamic measurement) By reducing, individual stability can be estimated. In some embodiments, the nearest neighbor interaction between dimers formed by the test sequence with the central hairpin is ignored in this calculation for convenience. For more accurate analysis, this interaction must be taken into account.

The core hairpin formed by has the following thermodynamic quantities:
ΔG = −5.3; ΔH = −37.8; ΔS = −104.8.

該オリゴヌクレオチド配列の2分子構造は、以下の工程を用いてmfold解析で検討する。
1.ボックス型mfold 配列において：

2.束縛性のボックス型において：
P 36 0 4
F 31 0 5
F 40 0 5
結果（1つを示す）：

二重鎖安定性を評価するために：

ヘアピン単独の熱力学的値を、完成した構造に関する値から減じる：

T_m（℃）＝ {ΔH ／ [ΔS + R ln（CT／4）]} − 273.15であり、Rがガス定数1.987（cal／Kmol）、lnが自然対数、およびCTが一本鎖の総モル濃度である。この計算式を用いた場合には、該構造の非ヘアピン部分T_mの計算値は46.1℃である。 The following probe sequence is used to illustrate this process.

The bimolecular structure of the oligonucleotide sequence is examined by mfold analysis using the following steps.
1. In a box-type mfold array:

2. In the bounding box type:
P 36 0 4
F 31 0 5
F 40 0 5
Results (show one):

To assess duplex stability:

Subtract the thermodynamic value of the hairpin alone from the value for the finished structure:

T _m (° C.) = {ΔH / [ΔS + R ln (CT / 4)]} − 273.15, R is a gas constant of 1.987 (cal / Kmol), ln is a natural logarithm, and CT is a single chain total Molar concentration. When using this formula, the calculated value of the non-hairpin portion the T _m of the structure is 46.1 ° C..

に対してのみ限定して利用されるものではなく、いずれの安定なヘアピン配列にも利用しうる。例えば、

しかしながら、異なるヘアピン配列を用いた場合には、mfoldを用いてその安定性を算出する必要があり、その熱力学量を以降の計算で利用する必要がある。 The above method is the core hairpin arrangement

It can be used for any stable hairpin sequence. For example,

However, when different hairpin sequences are used, it is necessary to calculate the stability using mfold, and it is necessary to use the thermodynamic quantity in subsequent calculations.

RNAStructure for predicting oligonucleotide interactions
Dimer formation can also be assessed using the RNAStructure program. Unlike mfold, RNAStructure can calculate all possible oligonucleotide-oligonucleotide interactions and provides an output .ct file. Second, visualize the structure using a .ct viewing program such as RNAStructure or RNAvis (1997, P. Rijk, University of Antwerp (UIA), available on the Internet at rma.uia.ac.be/rnavis) The stability of dimer formation is then evaluated using the nearest neighbor model (Borer et al., 1974) and DNA nearest neighbor parameters (Allawi & SantaLucia, 1997).

の性質を評価するために、二量体形成に関する「RNAStructure」のDNAフォールド・インターモレキュラー・モジュールを用いて、該配列をファイル（例えば、プローブ配列）に保存し、以下のパラメーターを設定する：
配列ファイル1:probe.seq
配列ファイル2:probe.seq
CTファイル：dimer.ct
最大％エネルギー差：50
最大構造数：20
ウインドウサイズ：変化させず
計算終了後、RNAStructureの「view」モジュールを用いて、得られた .ct ファイルを見ることができる。通常、該.ct ファイルには複数の構造が含まれる。「view」モジュールを用いて、個々のものを閲覧する。上記のように、RNAStructureに従い試験配列が形成しうる二量体の1つを以下に示す：

最近隣モデル（すなわち、DNA最近隣およびミスマッチ・パラメーター [Allawi & SantaLucia, 1997]を用いる）に従い、1MのNaClおよび100μMのプローブ濃度におけるこの二重鎖の安定性は、

である。
配列ファイル1および2を入れ替えることによって、RNAStructureを用いて、INVADERアッセイ反応に存在する総てのDNAオリゴヌクレオチド対の間における二量体形成の可能性を評価することができる。 For example, an array

In order to evaluate the nature of the sequence, the sequence is stored in a file (eg, probe sequence) using the “RNAStructure” DNA fold intermolecular module for dimer formation and the following parameters are set:
Sequence file 1: probe.seq
Sequence file 2: probe.seq
CT file: dimer.ct
Maximum% energy difference: 50
Maximum number of structures: 20
Window size: After completion of the calculation without change, you can view the resulting .ct file using the “view” module of RNAStructure. Typically, the .ct file contains multiple structures. Use the “view” module to view individual items. As described above, one of the dimers that the test sequence can form according to RNAStructure is shown below:

According to the nearest neighbor model (ie, using DNA nearest neighbor and mismatch parameters [Allawi & SantaLucia, 1997]), the stability of this duplex at 1 M NaCl and 100 μM probe concentration is

It is.
By exchanging sequence files 1 and 2, RNAStructure can be used to assess the possibility of dimer formation between all DNA oligonucleotide pairs present in the INVADER assay reaction.

iv. Assessment of assay performance Probe sets selected according to the above guidelines can be tested with the INVADER assay to assess their performance. Although oligonucleotides are designed to run at or near a specific desired reaction temperature, the best performance in a given design may not be accurately obtained at the desired temperature. Therefore, when evaluating a new INVADER assay probe set, it is useful to examine the performance of the INVADER assay at multiple different reaction temperatures, ranging from about 10 to 15 ° C., centered on the design temperature. For convenience, temperature optimization with a temperature gradient thermal cycler using a fixed amount of RNA (eg, 2.5 attomole in vitro transcript per reaction) and a fixed time (eg, 1 hour for each of the first and second reactions) Can be implemented. Thermal gradient testing provides the best performance of the designed probe set (e.g., the highest level of target-specific signal compared to the background signal usually results in multiple zero target background signals or “zero folding” Temperature) is revealed.

The results can be examined to determine how close the measured optimum temperature is to the set temperature in operation. In certain embodiments, it is preferred to include a probe set that operates at or near a preselected temperature. When the optimum temperature measured is higher than the desired reaction temperature, in a manner that can reduce the T _m of a probe / target (e.g., shortening least one base, or modified to contain one or more bases mismatched bases), The design of the probe can be changed. In embodiments where the stacker oligonucleotide is not utilized, the reaction temperature is greater than 7 ° C. compared to the desired reaction temperature, and performance (eg, zero folding) is acceptable, the probe • By using a 3 ′ mismatch in the oligonucleotide, the reaction temperature can be lowered without changing the performance of the assay.

  LOD can be determined by performing the reaction with different amounts of target RNA (eg, in vitro transcripts of known concentrations of control RNA). In a preferred embodiment, the LOD in the designed assay is less than 0.05 attomole. In a particularly preferred embodiment, the LOD is less than 0.01 attomole in the designed assay. The same guidelines as described above for reducing the LOD of the designed assay can be used to increase the LOD of the designed assay (ie, to be less sensitive to the presence of the target RNA). For example, it may be preferable to detect abundant and rare RNAs in the same reaction. In such reactions, it is preferable to attenuate the signal generated for abundant RNA so that it is not stronger than the signal from a rare species. In some embodiments, this can be achieved by designing a probe set that reduces signal generation (eg, at least 0.5 attomole (but not less) of LOD). In some embodiments, a single-step INVADER assay is used to detect abundant targets in a sample and, for non-abundant analytes in the same sample, a continuous amplification that amplifies the signal as described in Section II. Use the INVADER reaction. In a preferred embodiment, a single step and continuous INVADER assay reactions are performed in a single reaction for different analytes.

  In some embodiments, a time course reaction is performed that measures the accumulation of signal for a known amount of target in response to different lengths of time. This measurement can provide a linear range, that is, a range that allows accurate quantitative measurements using a given assay design with respect to time and starting level of target RNA.

v. Design and assay optimization Designed assays may not meet the above preferred performance criteria. Several variables of performance in INVADER assay reactions are described herein. These variables can be used alone or in combination to optimize the performance of the INVADER assay for the detection of RNA targets. For example, in some embodiments, stacker oligonucleotides are used. Without limiting the present invention to a particular mechanism of action, in some embodiments, stacker oligonucleotides improve assay performance by altering the hybridization properties (eg, T _m ) of a probe or INVADER oligonucleotide. be able to. In some embodiments, stacker oligonucleotides contribute to improved performance by allowing the use of shorter probes. In other embodiments, stacker oligonucleotides contribute to improved performance by changing the folding structure of the target nucleic acid. In yet another aspect, promoting the activity of a stacker oligonucleotide can include a combination of these and other mechanisms.

  In other embodiments, the target site can be moved. In some embodiments, the reaction is optimized by testing multiple probe sets that shift along sites suspected of being accessible. In preferred embodiments, such probe sets are shifted 1-2 base increments along the accessible site. In embodiments where accessible sites are predicted in advance only by computer analysis, physical detection of accessible sites is utilized to optimize probe set design. In a preferred embodiment, the RT-ROL method for detecting accessible sites is used. In some embodiments, optimization with respect to probe set design requires shifting the target site to a newly identified accessible site.

  For example, in an embodiment where accessible sites have been identified but probe set performance is poor, changing the probe 5 'arm design improves assay performance without changing the target site can do. In other embodiments, reducing the background signal by changing the length of the ARRESTOR oligonucleotide (eg, increasing the length of the portion complementary to the 5 ′ arm region of the probe) Can improve the performance.

Other variables in oligonucleotide design can also be utilized to alter assay performance. Certain modifications can be made to shift the ideal operating temperature of the probe set design to the preferred temperature range. For example, the use of shorter oligonucleotides and the introduction of mismatches usually lowers the T _m and lowers the ideal operating temperature of the designed oligonucleotide. Conversely, the use of longer oligonucleotides and the use of stacking oligonucleotides usually increases T _m and increases the ideal operating temperature of the designed oligonucleotide.

Other variables can be used to change other aspects of the performance of the oligonucleotide in the assay. For example, the enzymatic recognition of oligonucleotides can be altered by the use of base analogs or modified bases. In some embodiments, such modified bases can be used to protect certain regions of the oligonucleotide from cleavage by nucleases. In other embodiments, the use of modified bases can affect the ability to construct the cleavage structure of an oligonucleotide that is not at the cleavage position (eg, act as an INVADER oligonucleotide that allows cleavage of the probe). These modified bases are called “blocker” or “blocking” modifications. In some embodiments, the assay oligonucleotide comprises a 2′-O-methyl modification. In other embodiments, the assay oligonucleotide comprises a 3 ′ end modification (eg, NH ₂ , 3 ′ hexanol, 3 ′ phosphate, 3 ′ biotin).

  In yet another embodiment, the performance of the assay can be affected by changing the reaction components. For example, the concentration of oligonucleotide is changed. The concentration of oligonucleotide can affect several aspects of the reaction. Since the melting temperature of the complex is partly a function of the concentration of the complex component, the concentration variation of the oligonucleotide component can be used as an aspect of reaction optimization. In the method of the present invention, ARRESTOR oligonucleotides can be used to regulate the availability of the first probe oligonucleotide in the INVADER assay reaction. In some embodiments, ARRESTOR oligonucleotides can be eliminated. Other reaction components including enzyme concentration, salt and divalent ion concentration and divalent ion type can also be varied.

VI. Kits for Performing RNA INVADER Assays In certain embodiments, the present invention provides kits that include one or more of the components necessary to practice the present invention. For example, the invention provides kits for storing or delivering (eg, INVADER assay) the enzymes and / or reaction components of the invention required to perform a cleavage assay. The kit can include any components necessary or desired for the enzyme or assay, or all of them. Such elements include reagents themselves, buffers, control reagents (eg, tissue samples, target oligonucleotides as positive and negative controls, etc.), solid supports, labels, text and / or pictorial instructions, and products Examples thereof include, but are not limited to, information, inhibitors, labels and / or detection reagents, environmental control materials (for example, ice, desiccant, etc.) related to packaging. In some embodiments, the kit provides a subset of the required components, while the remaining components are user supplied specifications. In certain embodiments, the kit includes two or more different containers that include a subset of the components each container delivers. For example, a first container (eg, a box) contains an enzyme (eg, a suitable storage buffer and a structure-specific cleavage enzyme in a container), and a second box contains an oligonucleotide (eg, an INVADER oligo). Nucleotides, probe oligonucleotides, control target oligonucleotides, etc.). In some embodiments, one or more reaction components are provided in a pre-dispensed form (ie, pre-measured for use in one step of a method that does not require re-measurement or re-dispensing). . In some embodiments, selected reaction components are mixed and pre-dispensed together. In a preferred embodiment, pre-dispensed reaction components are provided in a pre-dispensed form in a reaction vessel (including but not limited to a reaction tube or, for example, a well of a microtiter plate). The In a particularly preferred embodiment, the reaction components dispensed in advance are dried on the bottom of the reaction vessel (for example, drying or lyophilization).

  Further, in some embodiments, the present invention provides a method of delivering a kit or reagent to a customer for use of the method of the present invention. The method of the present invention is not limited to a particular group of customers. Indeed, the method of the present invention has applications in providing kits or reagents to customers in many sectors of the biological and medical communities. Such customers include, but are not limited to, academic laboratory customers, biotech and medical industry customers, and government laboratory customers. The method of the present invention provides for all aspects of providing kits or reagents to customers, including but not limited to marketing, sales, delivery and academic.

  In some embodiments of the present invention, quality control (QC) and / or quality assurance (QA) experiments are performed prior to delivering kits or reagents to customers. Such QC and QA techniques typically involve testing reagents in experiments similar to industrial applications (eg, using assays similar to those described herein). Testing may include experiments to determine product retention and resistance to a wide range of solutions and / or reaction conditions (eg, temperature, pH, light, etc.).

  In certain embodiments of the present invention, prior to sale, the compositions and / or methods of the present invention are disclosed and / or presented to customers (e.g., prints or website advertisements, demos, etc.). Indicates use or function or component of the present invention. However, in some embodiments, the customer is not notified about the inclusion or use of one or more ingredients in the product being sold. In such embodiments, for example, not by knowledge of how and how it works (ie, the user does not need to know the components of the kit or reaction mixture), but the improvement of the product (eg, kit) Sales are promoted according to the desired and / or desired functions. Thus, the present invention creates kits, reagents or assay methods that are available to the user regardless of whether the user has knowledge of the components or mechanisms of the system.

  Accordingly, in one aspect, sales and marketing activities are to provide information regarding the new and / or improved properties of the methods and compositions of the present invention. In other embodiments, no information about such mechanisms is provided as marketing material. In some embodiments, information is collected from customers for the purpose of obtaining information regarding the type of assay component or delivery system that best meets the demand. Such information is useful in designing kit components and marketing activities.

VII. INVADER Assay for Direct Detection and Measurement of Specific RNA Samples Specific examples of mRNA detected or measured using the methods, compositions and systems of the present invention are shown below.

Housekeeping controls Typically, if a test sample contains a predictable or invariant amount of RNA, such RNA is a useful control target in detection assays. Such controls are useful in several ways, such as confirming that the assay is functioning properly and comparing or measuring the results of another RNA test, which can be used as a standard to help explain the results. However, it is not limited to these. The following gene mRNAs have particular uses in the methods of the invention.

Human ubiquitin and mouse / rat ubiquitin The ubiquitin system is the main pathway for selective proteolysis. Degradation by this system assists various cell functions such as DNA repair, cell cycle progression, signal transduction, transcription, and antigen presentation. The ubiquitin pathway also removes misfolded, misplaced, or other abnormal proteins. This pathway requires that ubiquitin (E1), a highly conserved 76 amino acid protein, be covalently bound to a specific lysine residue on the substrate protein.

Human, rat, and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
GAPDH is an important enzyme in glycolysis and gluconeogenesis pathways. This homotetrameric enzyme catalyzes the oxidative phosphorylation that converts D-glyceraldehyde-3-phosphoric to 1,3-diphosphoglycerate in the presence of cofactors and inorganic phosphate. Various diverse biological properties have been reported for GAPDH. These include functions such as endocytosis, mRNA regulation, tRNA export, DNA replication, DNA repair and neuronal apoptosis.

Cytokines A growing family of regulatory proteins has been identified that transmit signals between cells of the immune system. These proteins, called cytokines, are known to control hematopoietic and immune system cell proliferation and differentiation, as well as physiological activity. Cytokines exhibit a wide range of biological activities against target cells derived from bone marrow, peripheral blood, fetal liver and other lymphoid or hematopoietic organs. The present invention describes a method of detecting cytokine expression. Such cytokines include, but are not limited to, exemplary members of the following cytokine family.

Human oncostatin M
Oncostatin M is a secreted polypeptide cytokine that consists of a single chain that controls the growth of certain tumor-derived and normal cell lines. Several types of cells have been shown to bind to oncostatin M protein. It has been shown to inhibit the growth of multiple types of tumor cells, but has also been shown to stimulate the growth of Kaposi's sarcoma cells.

Human transforming growth factor β (TGF-β)
Transforming growth factor beta (TGF-beta) is a member of a family of structurally related cytokines that trigger a variety of responses including proliferation, differentiation and morphogenesis in a variety of different cell types. In vertebrates, at least five different TGF-β types have been identified, referred to as TGF-β1 to TGF-β5. They have a high degree of amino acid sequence identity (60-80%). Although TGF-β1 was first characterized as capable of inducing anchorage-independent growth in normal rat kidney cells, the effect on many types of cells is mitotic. This shows a strong growth inhibitory effect on many types of cells including normal and transformed epithelium, endothelium, fibroblasts, nerve, lymphoid and hematopoietic cells. In addition, TGF-β plays a central role in the control of extracellular matrix formation and cell-matrix adhesion processes.

Human monocyte chemoattractant protein-1 (MCP-1)
A group of emerging chemotactic cytokines, also called chemokines or interclines, has been identified as belonging to this family of cytokines. As two subfamilies of chemokines, α and β are known based on the position on the chromosome and the composition of cysteine residues.

  The human gene encoding the β subfamily protein is located on chromosome 17 (the mouse counterpart gene is clustered on mouse chromosome 11; this chromosome corresponds to human chromosome 17). To do. Homology in subfamilies ranges from 28-45% within species and 22-55% between species. An example of a member is MCP-1, which is a human protein (monocyte chemoattractant protein-1). MCP-1 has multiple effects specific to monocytes. It is a potent chemoattractant for human monocytes in vitro, promoting free cytoplasmic free calcium and monocyte respiratory burst. MCP-1 has been reported to activate monocyte-mediated antitumor activity and induce tumor damage activity. MCP-1 is known to be an important factor mediating monocyte infiltration in tissue inflammatory processes such as rheumatoid arthritis and alveolitis. This factor plays a fundamental role in the recruitment of monocytes-macrophages to progressive atherosclerotic lesions.

Human tumor necrosis factor α (TNF-α)
Tumor necrosis factor α (TNF-α, also called cachectin) is an important cytokine that plays a role in host defense. Cytokines are produced primarily by macrophages and monocytes in response to infection, invasion, injury or inflammation. Examples of inducers of TNF-α include bacterial endotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 and IFN-γ.

  Despite the protective effects of cytokines, overexpression of TNF-α often results in disease states, particularly infectious, inflammatory and autoimmune diseases. This step may involve an apoptosis pathway. Infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria and ADIS; rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and High levels of plasma TNF-α in autoimmune diseases such as transplant rejection (allogeneic transplantation); and organ failure states such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction Are known. Other diseases involving TNF-α include asthma, ischemic brain injury, non-insulin-dependent diabetes, insulin-dependent diabetes, hepatitis, atopic dermatitis and pancreatitis. Furthermore, it has been suggested that TNF-α inhibitors are useful for the prevention of cancer. Increased TNF-α expression also plays a role in obesity. It is known that TNF-α is expressed in human adipocytes, and its expression is usually increased in correlation with obesity.

Human interleukin-6 (IL-6)
IL-6 is the standard name for cytokines called B lymphocyte differentiation factor, interferon β2, 26Kd protein, hybridoma / plasma cell tumor growth factor, hepatocyte stimulating factor and the like.

  IL-6 induces activated B cells to differentiate into antibody producing cells. With respect to T cells, IL-6 induces IL-2 receptor expression on specific T cell lines or thymocytes, where mitogen-stimulated T cells produce IL-2. With respect to hematopoietic cells, IL-6 cooperatively induces hematopoietic stem cell proliferation in the presence of IL-3. Furthermore, recently it has been reported that IL-6 acts like a thrombopoietin.

  Various cells produce IL-6. Lymphocytes produce IL-6, and human fibroblasts stimulated with poly (I) -poly (C) and cycloheximide produce IL-6. Mouse cells stimulated with poly (A) -poly (U) produce murine IL-6. Stimulation inducers vary and include known cytokines such as IL-1, TNF and INF-β, growth factors such as PDGF and TGF-β, LPS, PMA, PHA, cholera toxin and the like. Furthermore, it has been reported that human vascular endothelial cells, macrophages, human oligoblastoma and the like also produce IL-6. It is also known that productivity can be further promoted by stimulating cells with inducers and then treating the cells with metabolic inhibitors such as verapamil, cycloheximide or actinomycin D. .

Human interleukin 1β (IL-1β)
Interleukin-1 (IL-1) is important for T and B lymphocyte activation and mediates many inflammatory processes. Two different molecular species of IL-1 have been isolated and expressed. These are called IL-1β and IL-1α. IL-1β is the major form at both the mRNA and protein levels produced by human monocytes. In these two human IL-1 molecular species, the amino acid homology is only 26%. Despite the different polypeptide sequences, the two molecular species of IL-1 are structurally similar, limiting amino acid homology to distant regions of the IL-1 molecule. In addition, two molecular species IL-1 induces fever, slow wave sleep and neutropenia, T and B lymphocyte activation, fibroblast proliferation, cytotoxicity against certain cells, collagenase Has the same biological properties including induction, hepatic acute phase protein synthesis and increased colony stimulating factor and collagen production. IL-1 also activates endothelial cells, increases leukocyte adhesion, PGI ₂ and PGE ₂ (prostaglandin) release, and inhibits platelet activating factor, procoagulant activator and plasminogen activator Facilitates the synthesis of factors. Clearly, IL-1 plays a central role in local and systemic host responses. Since many of the biological effects of IL-1 in vivo occur at picomolar (pg) concentrations, IL-1 production appears to have the basic features of host defense mechanisms.

Human interleukin 2 (IL-2)
Interleukin-2 (IL-2) is the main growth factor for T lymphocytes. By controlling the activity of helper T lymphocytes, IL-2 promotes humoral and cellular immune responses. By stimulating cytotoxic CD8 T cells and NK cells, this cytokine is involved in defense mechanisms against tumors and viral infections. IL-2 is used to treat metastatic melanoma and renal adenocarcinoma. IL-2 is used in clinical trials for many types of cancer. It is also used in patients infected with HIV and significantly increases the total number of CD4. Human IL-2 is a protein containing 133 amino acids (aa) and has a three-dimensional structure in which four α-helices are connected by loops of various lengths. IL-2R is composed of three chains: α, β and γ. IL-2Rα regulates the affinity of the receptors IL-2Rβ and IL-2Rγ and is important for IL-2 signal transduction. Different molecular regions of IL-2 that interact with the three chains of IL-2R have been determined. More specifically, it has been found that the NH ₂ terminal region of IL-2 (residues 1-30) and helix A control the IL-2 / IL-2Rβ interaction.

Human interleukin 8 (IL-8)
Human IL-8 is a cytokine with various names such as neutrophil activating protein, neutrophil migration factor (NCF) and T cell migration factor. Multiple types of cells secrete IL-8 in response to appropriate stimuli. Activated monocytes and macrophages and fetal fibroblasts secrete IL-8.

  IL-8 is known to induce neutrophil migration and activate functions such as neutrophil degranulation, superoxide ion release and adhesion to monolayer endothelial cells. There are several known conditions for conditions involving infiltration of leukocytes into the lesion. Examples include pulmonary cystic fibrosis, idiopathic pulmonary fibrosis, pulmonary diseases such as adult respiratory distress syndrome, sarcoidosis and empyema; skin diseases such as psoriasis; rheumatoid arthritis; and inflammatory bowel disease (Crohn's disease) .

  The amino acid sequence of human IL-8 is described in International Publication No. 89/08665 by Matsushima et al. More recently, monocyte-derived IL-8 has undergone various steps at the N-terminus, and Matsushima et al.'S first disclosed IL-8 is actually a factor with an additional 7 or 5 amino acids at the N-terminus. (Yoshimura et al., Mol Immunol 26:87 [1989]). The breakdown of total monocyte-derived IL-8 is about 8% for the longest molecular species, about 47% for the next longest molecular species, and about 45% for the shortest molecular species.

Human interleukin 10 (IL-10)
Interleukin 10 (IL-10), a recently discovered lymphokine, was originally described as an inhibitor of interferon-gamma synthesis and is thought to be the main mediator of humoral immune responses . The two types of immune responses that are often mutually exclusive are humoral (antibody-mediated) and delayed-type hypersensitivity.

  These two different immune responses are thought to be caused by two types of helper T cell clones. That is, Th1 and Th2 helper T cells, which show different cytokine secretion patterns. Mouse Th1 cell clones secrete interferon-γ and IL-2 and selectively induce delayed hypersensitivity reactions. Th2 cell clones secrete IL-4, IL-5 and IL-10 and aid in humoral responses. The reason for the contrast in the immune response is that interferon-γ secreted by Th1 cell clones inhibits Th2 clone growth in vitro, and IL-10 secreted by Th2 cell clones inhibits Th1 cell clones inhibit cytokine secretion . Thus, the two types of T helper cells are mutually inhibitory and provide a basis for two contrasting immune responses.

  IL-10 has been cloned and sequenced from both mouse and human T cells. Both sequences contain an open reading frame encoding a 178 amino acid polypeptide, the N-terminus contains an 18 amino acid hydrophobic leader sequence, and the amino acid sequence homology is 73%.

Human interleukin 4 (IL-4)
Interleukin-4 (IL-4, also known as B cell stimulator or BSF-1) originally has the ability to stimulate B cell proliferation in response to low concentrations of antibodies to surface immunoglobulins. Characterized as having. More recently, IL-4 has been shown to have a wide range of biological activities including proliferation costimulation of T cells, mast cells, granule cells, megakaryocytes and erythrocytes. In addition, IL-4 stimulates proliferation of multiple IL-2 and IL-3 dependent cell lines, induces expression of class II major histocompatibility complex molecules on resting B cells, and stimulates B cells Promotes secretion of IgE and IgG1 isoforms from Mouse and human IL-4 have been clearly characterized by recombinant DNA technology and homogenous purification of native mouse proteins.

  Specific cell surface receptors for IL-4 expressed in primary cells and in vitro cells from mammals mediate IL-4 biological activity. IL-4 binds to the receptor and further transmits biological signals to various immune effector cells.

Human interferon gamma (IFN-γ)
Like interleukins, interferons belong to the class of cytokines and various types exist. That is, interferon-α, interferon-β, interferon-γ, interferon-ω, interferon-τ, and the like. Interferon-γ is a glycoprotein whose amino acid sequence was revealed in 1982. Under mature conditions, interferon-γ has 143 amino acids and a molecular weight of 63 to 73 kilodaltons.

  The tertiary and quaternary structure of the non-glycosylated protein was revealed in 1991. According to this, interferon-γ exists as a homodimer, and the monomers are oriented in opposite directions so that the C-terminal of one monomer is located near the N-terminal of the other monomer. ing. Each of these monomers has six α-helices. Interferon gamma is also called immune interferon because it has non-specific antiviral, antiproliferative and particularly immunomodulatory effects. Its production in T helper-lymphocytes is stimulated by mitogens and antigens. The effect of expressed interferon-γ has not been clarified yet, but intensive studies are underway. In particular, interferon-γ induces macrophage activation and class 2 histocompatibility antigen synthesis. In vitro, interferon-γ activity is usually examined by reducing the virus-induced cytopathic effect of interferon-γ treatment. Since it has non-antigen-specific antiviral, antiproliferative and immunomodulatory activities, it is suitable as a therapeutic agent for humans, for example, kidney tumors and chronic granulomatous diseases.

Cytochrome P450
The term cytochrome P-450 represents the family of enzymes most important for the metabolism of xenobiotics such as drugs, carcinogens, and environmental chemicals, and multiple classes of endobatics such as steroids and prostaglandins ( Located on the endoplasmic reticulum, the concentration of the protein is high in cells of the liver and small intestine). The levels of members of the cytochrome P450 family vary, and their expression and activity are controlled by variables such as chemical environment, sex, developmental stage, nutrition, and age.

  More than 200 cytochrome P450 genes have been identified. There are multiple molecular species in these P450 genes, each showing specificity for an individual chemical in the class of compounds described above. It may be a drug or a carcinogen, but the substrate may be metabolized by two or more cytochrome P450s. Cytochrome P450 genetic polymorphisms result in traitally distinct subpopulations that differ in the biotransformability of certain drugs and other chemical compounds.

  The present invention provides a method for detecting cytochrome P450 mRNA. Such cytochromes include human CYP 1A1, human CYP 1A2, human CYP 2B1, human CYP 2B2, human CYP 2B6, human CYP 2C19, human CYP 2C9, human CYP 2D6, human CYP 3A4, human CYP 3A5, human CYP 3A7. Rat CYP 2E1, rat CYP 3A1, rat CYP 3A2, rat CYP 4A1, rat CYP 4A2, and rat CYP 4A3, but are not limited thereto.

  The following examples illustrate certain preferred embodiments and aspects of the invention, but are not intended to limit the scope of the invention.

In this disclosure, the following abbreviations are used. Afu (Archaeoglobus fulgidus); Mth {Methanobacterium thermoautotrophicum}; Mja (Methanococcus jannaschii); Pfu (Pyrococcus furiosus (ios) ); Pwo (Pyrococcus woesei); Taq (Thermus aquaticus); Taq DNAP, DNAP Taq, and Taq Pol I (Thermus aquaticus DNA polymerase I); DNAPStf (DNA polymerase Stofel fragment); DNAPEcl (DNA polymerase I of E. coli); Tth (Thermus thermophilus); Ex. (Example); Fig. (Figure); ° C (Celsius); g ( (Gravity field); hr (hour); min (minute); oligo (oligonucleot ide)); rxn (reaction); vol (volume); w / v (weight to volume); v / v (volume to volume); BSA (bovine serum albumin); CTAB (cetyltrimethyl bromide) Ammonium); HPLC (high performance liquid chromatography); DNA (deoxyribonucleic acid); p (plasmid); μl (microliter); ml (milliliter); μg (microgram); mg (milligram); M (mole); (Mmol); μM (micromol); pmoles (picomoles); amoles (atomoles); zmoles (zeptomoles); nm (nanometers); kdal (kilodaltons); OD (optical density); EDTA (ethylenediaminetetraacetic acid); FITC (fluorescein isothiocyanate); SDS (sodium dodecyl sulfate); NaPO ₄ (sodium phosphate); NP-40 (Nonidet P-40); tris (tris (hydroxymethyl) - aminomethane PMSF (phenylmethylsulfonyl fluoride); TBE (tris-borate-EDTA, ie containing EDTA in a tris buffer titrated with boric acid instead of HCl); PBS (phosphate buffered saline); PPBS (1 mM) Phosphate buffered saline containing PMSF); PAGE (polyacrylamide gel electrophoresis); Tween (polyoxyethylene-sorbitan); ATCC (American Type Culture Collection, Rockville, MD); Coriell (Coriell Cell Repositories, Camden, NJ); DSMZ (Deutsche Sammlung von Mikroorganismen und Zellculturen, Braunschweig, Germany); Ambion (Ambion, Inc., Austin, TX); Boehringer (Boehringer Mannheim Biochemical, Indianapolis, IN); MJ Research (MJ Research, Watertown, MA); Sigma (Sigma Chemical Company, St. Louis, MO); Dynal (Dynal AS, Oslo, Norway); Gull (Gull Laboratories, Salt Lake City, UT); Epicentre (Epicentre Technolog Lampies (Biological Labs., Inc., Coopersberg, PA); MJ Research (MJ Research, Watertown. MA); National Biosciences (National Biosciences, Plymouth, MN); NEB (New England Biolabs, Beverly) , MA; Novagen (Novagen, Inc., Madison, WI); Promega (Promega, Corp., Madison, WT); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Clonetech (Clonetech, Palo Alto, CA); Pharmacia (Pharmacia, Piscataway, NJ); Milton Roy (Milton Roy, Rochester, NY); Amersham (Amersham International, Chicago, IL); and USB (US Biochemical, Cleveland, OH); Glen Research (Glen Research, Sterling, VA) Coriell (Coriell Cell Repositories, Camden, NJ); Gentra (Centra, Minneapolis, MN); Third Wave Technologies (Third Wave Technologies, Madison, Wis.); PerSeptive Biosystems (PerSeptive Biosystems, Framington, MA); Microsoft (Microsoft, Redmond); , WA); Qiagen (Qiagen, Valencia, CA); Molecular Probes (Molecular Probes, Eugene, OR) ; VWR (VWR Scientific); Advanced Biotechnologies (Advanced Biotechnologies, INC, Columbia, MD.); And Perkin Elmer (PE Biosytems and Applied Biosystems, Foster City, also known as CA).

Example 1
Rapid colony screening for 5 'nuclease activity Natural 5' nucleases and the enzymes of the present invention can be tested directly for various functions. These include, but are not limited to, 5 'nuclease activity against RNA or DNA targets, and background specificity using other substrates that are representative of structures that may be present in the target detection reaction. It is not a thing. Examples of nucleic acid molecules having suitable test structures are shown schematically in FIGS. 18A-D and FIGS. 21-24. The following screening techniques were developed to characterize 5 'nucleases quickly and efficiently, and to determine whether new 5' nucleases have improved or desired activity. Enzymes that show improved cycling rates for RNA or DNA targets, or that have reduced target-independent cleavage, are worth investigating in more detail. In general, modified proteins obtained by random mutagenesis were tested by rapid colony screening using the substrates shown in FIGS. 18A and 18B. Next, rapid protein extraction was performed, and an activity test on another structure (for example, shown in FIGS. 18C to D) was performed using the protein extract. Initial screening or subsequent screening and characterization for improved activity of the enzyme was performed using other cleavage complexes as illustrated in FIGS. The particular sequence used to form such a test cutting structure is not intended to limit the scope of the present invention. A person skilled in the art can understand how to design and produce a similar nucleic acid that forms a similar structure for rapid screening.

  By testing in this order, the total number of tests can be reduced, saving time and reagents. The present invention is not limited by the order of tests for testing enzyme function. Next, mutants that exhibit a reasonable cycling rate against the RNA or DNA target are cultured overnight to perform rapid protein extraction. Alternatively, a subset or all of the cutting tests can be performed simultaneously.

  For convenience, individual types of rapid screening can also be performed on separate microtiter plates. For example, one plate is prepared for the RNA INVADER activity test, and one plate is prepared for the DNA INVADER activity test. About 90 different colonies can be screened on one plate. Screened colonies are derived from original (natural) 5 'nuclease clones, mutagenesis reactions (eg, many colonies from a single plate) or various reactions (colonies selected from multiple plates), etc. It may be derived from various origins.

  Ideally, the same preparation reagents are used and the positive and negative controls are run as variants on the same plate. Examples of good positive controls include colonies containing unmodified enzymes, or enzymes that have been previously modified to compare activity with new mutants. For example, if a mutagenesis reaction is performed on the Taq DN RX HT construct (below), the unmodified Taq DN RX HT construct is selected as a standard to compare the effect of mutagenesis on enzyme activity. Another control enzyme can also be incorporated into the rapid screening test. For example, Tth DN RX HT (below; unless otherwise specified, TaqPol enzyme and TthPol enzyme described below represent DN RX HT derivatives) can be incorporated with Taq DN RX HT as a standard for enzyme activity. This allows the modified enzyme to be compared to two known enzymes with different activities. Negative controls should also be performed to examine background reaction levels (ie, cleavage or probe degradation by sources other than the nuclease being compared). Good negative control colonies include those containing only the vector used for cloning and mutagenesis, eg, those containing only the pTrc99A vector.

Two factors that affect the number of colonies selected in the specific mutagenesis reaction of the initial rapid screen include the following:
(1) Total number of colonies obtained by mutagenesis reaction; (2) Whether the mutagenesis reaction is site-specific or randomly distributed over the entire gene or gene region. For example, if there are at most 5-10 colonies present on the plate, it is easy to test all colonies. If there are hundreds of colonies, these subsets can also be analyzed. Typically, 10-20 colonies are tested for site-directed mutagenesis reactions, and typically 80-100 or more colonies are tested for single random mutagenesis reactions.

  As described, the modified 5 ′ nucleases described in these experimental examples were tested in the manner detailed below.

1μM INVADERオリゴ（配列番号：224）

および4nM RNA標的（配列番号：225）

を含む。5μlの2x基質混合物を96ウェルマイクロタイタープレートの試料用ウェルの各々に分注した（ロープロフィール MULTIPLATE 96, M.J. Research, Inc.）。 A. Rapid screening; INVADER activity against RNA targets (Figure 18A)
A 2x substrate mixture was prepared. This is 20 mM MOPS, pH 7.5, 10 mM MgSO ₄ , 200 mM KCl, 2 μM FRET-probe oligo (SEQ ID NO: 223)

1 μM INVADER oligo (SEQ ID NO: 224)

And 4 nM RNA target (SEQ ID NO: 225)

including. 5 μl of 2 × substrate mixture was dispensed into each of the sample wells of a 96 well microtiter plate (low profile MULTIPLATE 96, MJ Research, Inc.).

  Single colonies (mutant, positive control and negative control colonies) were picked and cell suspensions were prepared. Each was suspended in 20 μl of water. It can be carried out conveniently at 1 well per colony using a 96 well microtiter plate.

Place 5 μl of cell suspension in appropriate test wells so that the final reaction conditions are 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl, 1 μM FRET-probe oligo, 0.5 μM INVADER oligo, and 2 nM RNA target. Added. The wells were covered with 10 μl of clear CHILLOUT 14 (MJ Research, Inc.) liquid wax and the samples were heated at 85 ° C. for 3 minutes and then incubated at 59 ° C. for 1 hour. After incubation, the plate was measured using a Cytofluor fluorescent plate reader with the following parameters: excitation 485/20, emission 530/30.

1μM INVADERオリゴ（配列番号：224）

1nM DNA標的（配列番号：226）

を含む。5μlの2x基質混合物を96ウェルマイクロタイタープレートの試料用ウェルの各々に分注した（MJ ロープロフィール）。 B. Rapid screening; INVADER activity against DNA targets (Figure 18B)
A 2x substrate mixture was prepared. This is 20 mM MOPS, pH 7.5, 10 mM MgSO ₄ , 200 mM KCl, 2 μM FRET-probe oligo (SEQ ID NO: 223)

1 μM INVADER oligo (SEQ ID NO: 224)

1 nM DNA target (SEQ ID NO: 226)

including. 5 μl of 2 × substrate mixture was dispensed into each of the sample wells of a 96 well microtiter plate (MJ low profile).

  Single colonies (mutant, positive control and negative control colonies) were picked and cell suspensions were prepared. Usually, 96-well microtiter plates were used and each was suspended in 20 μl of water.

Add 5 μl of cell suspension to appropriate test wells so that the final reaction conditions are 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl, 1 μM FRET-probe oligo, 0.5 μM INVADER oligo, and 0.5 nM DNA target. Added to. The wells were covered with 10 μl of clear CHILLOUT 14 (MJ Research, Inc.) liquid wax and the sample was heated at 85 ° C. for 3 minutes and then incubated at 59 ° C. for 1 hour. After incubation, the plate was measured using a Cytofluor fluorescent plate reader with the following parameters: excitation 485/20, emission 530/30, gain 40, 10 readings per well.

C. Rapid protein extraction (crude cell lysate)
The mutants that gave positive or unpredictable results in the RNA or DNA INVADER assay were further analyzed. Specifically, analysis was performed on background activity against the X structure or hairpin substrate (FIGS. 18C and D, respectively). As described above, rapid colony screening can be utilized. Testing for background or abnormal enzyme activity can be performed by simply changing the substrate. In another method, positive clones are grown in small scale overnight, rapid protein extraction is performed, and the crude lysate of this cell is tested for another protein function. An example of a rapid protein extraction method that can be used is described in detail below. Inoculate 2-5 ml of LB (containing appropriate antibiotics for selection of plasmid; see, eg, Maniatis volumes 1-3) with the remaining volume of 20 μl water-cell suspension, 37 Incubated overnight at ° C. Approximately 1.4 ml of the culture was transferred to a 1.5 ml microcentrifuge tube and microcentrifuged at room temperature for 3-5 minutes at maximum speed (eg, 14,000 rpm in an Eppendorf 5417 tabletop microcentrifuge) to pellet the cells. The supernatant was removed and the cell pellet was suspended in 100 μl of TES buffer (pH 7.5) (Sigma). Lysozyme (Promega) was added at a final concentration of 0.5 μg / μl and the sample was incubated at room temperature for 30 minutes. The sample was then heated at 70 ° C. for 10 minutes to inactivate lysozyme. Microcentrifuge for 5 minutes at maximum speed to pellet the cell debris. The supernatant was removed, and the crude lysate of the cells was used in the following enzyme activity assay.

および0.5nMのDNA標的（配列番号：226）であった。ウェルを10μlの透明なCHILLOUT 14（M.J. Research, Inc.） 液体ワックスで覆い、反応液を85℃3分加熱し、次に59℃で1時間保温した。保温後、Cytofluor蛍光プレート読み取り装置を用い、以下のパラメータでプレートの測定を行った：励起485／20、発光530／30、ゲイン40、ウェル当たりの読みが10。 D. Rapid Screening: Background Specific X Structure Substrate (Figure 18C)
The reaction was carried out under the conditions detailed above. 1 μl of crude cell lysate was added to 9 μl of reaction components to a final volume of 10 μl. Final concentrations are 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl, 1 μM FRET-probe oligo (SEQ ID NO: 223), 0.5 μM X structure INVADER oligo (SEQ ID NO: 227)

And 0.5 nM DNA target (SEQ ID NO: 226). The wells were covered with 10 μl of clear CHILLOUT 14 (MJ Research, Inc.) liquid wax and the reaction was heated at 85 ° C. for 3 minutes and then incubated at 59 ° C. for 1 hour. After incubation, the plate was measured using a Cytofluor fluorescent plate reader with the following parameters: excitation 485/20, emission 530/30, gain 40, 10 readings per well.

E. Rapid Screening: Background Specific Hairpin Substrate (Figure 18D)
The reaction was carried out under the conditions detailed above. 1 μl of crude cell lysate was added to 9 μl of reaction components to a final volume of 10 μl. The final concentration was 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl, 1 μM FRET-probe oligonucleotide (SEQ ID NO: 223), and 0.5 nM DNA target (SEQ ID NO: 226). The wells were covered with 10 μl of clear CHILLOUT 14 (MJ Research, Inc.) liquid wax and the reaction was heated at 85 ° C. for 3 minutes and then incubated at 59 ° C. for 1 hour. After incubation for 1 hour, the plate was measured using a Cytofluor fluorescent plate reader with the following parameters: excitation 485/20, emission 530/30, gain 40, 10 readings per well.

F. Activity measurement using IrTI and IdT targets (Figure 24)
The 5 ′ nuclease activity assay was performed in 10 μl reaction containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10 μg / ml tRNA, 100 mM KCl, and 5 mM MgSO ₄ . The probe concentration (SEQ ID NO: 167) was 2 mM. Substrate (final concentrations of IrTI (SEQ ID NO: 228) or IdT (SEQ ID NO: 229) were 10 nM or 1 nM, respectively), and about 20 ng of the enzyme prepared by the method of Example 3 were added to the above reaction buffer. Mixed with liquid and overlaid with CHILLOUT (MJ Research) liquid wax. The reaction was brought to a reaction temperature of 57 ° C. and started by adding MgSO ₄ and incubated for 10 minutes. The reaction was then stopped by adding 10 μl of 95% formamide containing 10 mM EDTA and 0.02% methyl violet (Sigma). Immediately before electrophoresis on a 20% denaturing acrylamide gel (cross-linked 19: 1) in a buffer containing 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA containing 7 M urea, the sample was at 90 ° C. for 1 min. Heated. Unless otherwise noted, 1 μl of stop reaction was added per lane. The gel was then scanned on a FMBIO-100 fluorescent gel scanner (Hitachi) using a 505 nm filter. FMBIO analysis software (version 6.0 (Hitachi)) was used to determine the fraction of the cleaved product from the intensity of the band that was uncut and matched to the cleaved substrate. Since the fraction of cleavage products did not exceed 20%, an approximate initial cut rate could be measured. The turnover rate was defined as the number of cleavage signal probes generated per minute per target molecule under these reaction conditions (1 / min).

G. Activity measurement with X structure (X) and hairpin (HP) targets (Figure 22)
Assay of 5 ′ nuclease activity was performed in 10 μl reaction containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10 μg / ml tRNA, 100 mM KCl, and 5 mM MgSO ₄ . Each oligo for hairpin structure formation (22A, SEQ ID NO: 230 and 231) or X structure formation (22B, SEQ ID NO: 230-232) was added at a final concentration of 1 μm. About 20 ng of the test enzyme prepared by the method of Example 3 was mixed with the above reaction buffer and overlaid with CHILLOUT (MJ Research) liquid wax. The reaction was brought to a reaction temperature of 60 ° C. and started by adding MgSO ₄ and incubated for 10 minutes. The reaction was then stopped by adding 10 μl of 95% formamide containing 10 mM EDTA and 0.02% methyl violet (Sigma). Immediately before electrophoresis on a 20% denaturing acrylamide gel (cross-linked 19: 1) in a buffer containing 45 mM Tris-borate, pH 8.3, 1.4 mM EDTA containing 7 M urea, the sample was run at 90 ° C. for 1 min. Heated. Unless otherwise noted, 1 μl of stop reaction was added per lane. The gel was then scanned on a FMBIO-100 fluorescent gel scanner (Hitachi) using a 505 nm filter. FMBIO analysis software (version 6.0 (Hitachi)) was used to determine the fraction of the cleaved product from the intensity of the band that was uncut and matched to the cleaved substrate. Since the fraction of cleavage products did not exceed 20%, an approximate initial cut rate could be measured. The turnover rate was defined as the number of cleavage signal probes generated per minute per target molecule under these reaction conditions (1 / min).

H. Activity measurement with human IL-6 target (Figure 10)
Assay of 5 ′ nuclease activity was performed in 10 μl reaction containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10 μg / ml tRNA, 100 mM KCl, and 5 mM MgSO ₄ . Reaction solution containing IL-6 DNA substrate, 0.05 nM IL-6 DNA target (SEQ ID NO: 163), each 1 μM probe (SEQ ID NO: 162) and INVADER (SEQ ID NO: 161) oligonucleotide, 60 ° C. for 30 minutes Reacted. A reaction containing the IL-6 RNA target (SEQ ID NO: 160) was performed under the same conditions except that the concentration of IL-6 RNA target was 1 nM and the reaction was performed at 57 ° C. for 60 minutes. . Each reaction contained approximately 20 ng of test enzyme prepared by the method of Example 3.

I. Activity measurement by synthetic target of r25mer (Fig. 23)
Reactions containing synthetic r25mer target (SEQ ID NO: 233) were performed under the same conditions (10 mM MOPS, except that the concentration of r25mer target was 5 nM and the reaction was performed at 58 ° C. for 60 minutes. 1 μM probe (SEQ ID NO: 234) and INVADER (SEQ ID NO: 235) oligos under pH 7.5, 0.05% Tween 20, 0.05% Nonidet P-40, 10 μg / ml tRNA, 100 mM KCl and 5 mM MgSO ₄ ) Performed in the presence of nucleotides. About 20 ng of each test enzyme was added to the reaction. The enzyme was prepared by the method of Example 3.

  All of the above tests can be modified to obtain optimal conditions for enzyme activity. For example, enzyme titration can be performed to determine the optimal enzyme concentration that yields maximum cleavage activity and minimum background signal. By way of example and not limitation, a number of mutant enzymes were tested in amounts of 10 ng, 20 ng and 40 ng. Similarly, temperature titration can be used for this test. By modifying the protein structure, the temperature requirements can be changed, so that the temperature range can be tested to identify the optimal conditions for the variant in question.

  The results obtained by such screening (using about 20 ng of mutant enzyme) are shown in Tables 3-8 and FIGS. 12, 14, 15, 19, and 25.

Example 2
Cloning and expression of DNA polymerase and mutant polymerase 5 'nucleases.
A. DNA polymerase from Thermus aquaticus and Thermos thermophilus
1. Cloning TaqPol and TthPol Type A DNA polymerase from Thermus genus bacteria has very high protein sequence identity (90% in the polymerization domain using the Lipman-Pearson method of DNA analysis software of DNAStar, WI) ), Similar behavior in both the polymerization assay and the nuclease assay. Therefore, as a representative of this class, the DNA polymerase genes of Thermus aquaticus (TaqPol), Thermus thermophilus (TthPol), and Thermus scottodactus were used. Other eubacterial polymerase genes include Escherichia coli, Streptococcus pneumoniae, Mycobacterium smegmatis, Thermus thermophilus, Thermos species, Thermotoga maritima, Thermotoga maritima, Thermotoga maritima, Thermotoga maritima, Thermotoga maritima, Thermotoga maritima These include, but are not limited to, Thermosipho africanus and Bacillus stearothermophilus. These are all suitable.

a. Initial isolation of TaqPol: mutant TaqA / G
The Taq DNA polymerase gene was amplified by polymerase chain reaction from the genomic DNA of Thermus aquaticus YT-1 strain (Lawyer et al., Supra) using the oligonucleotides set forth in SEQ ID NOs: 236 and 237 as primers. The obtained DNA fragment has a recognition sequence for the restriction endonuclease EcoRI at the 5 ′ end of the coding sequence and a BglII sequence at the 3 ′ end of the coding sequence. Cleavage with BglII results in a 5 ′ overhang or “sticky end” that matches the end obtained with BamHI. DNA amplified by PCR was digested with EcoRI and BamHI. A 2512 bp fragment containing the coding region of the polymerase gene was purified by gel and then ligated to a plasmid containing an inducible promoter.

  In one embodiment of the present invention, a pTTQ18 vector containing a hybrid trp-lac (tac) promoter was used (M.J.R. Stark, Gene 5: 255 [1987]). The tac promoter is under the control of the E. coli lac repressor protein. By applying suppression, the synthesis of the gene product is suppressed until the bacterial growth reaches the desired level, at which point a specific inducer, isopropyl-β-D-thiogalactopyranoside (IPTG) is added To release the suppression. By such a system, the expression of an exogenous protein that slows or inhibits the growth of the transformant can be controlled.

  When a particularly strong bacterial promoter such as synthetic Ptac is present on a multicopy plasmid, it cannot be properly suppressed. When highly toxic proteins are placed under the control of such promoters, small amounts of leakage occur even in the absence of inducers, which can be detrimental to bacteria. In another embodiment of the present invention, another option is conceivable that suppresses the synthesis of the cloned gene product. The cloned mutant Taq polymerase gene is expressed using the non-bacterial promoter of bacteriophage T7 contained in the plasmid vector series pET-3 (Studier and Moffatt, J. Mol. Biol., 189: 113). [1986]). This promoter initiates transcription only by T7 RNA polymerase. In suitable strains such as BL21 (DE3) pLYS, the phage T7 RNA polymerase gene is contained in the bacterial genome and is under the control of the lac operator. The advantage of such a configuration is that the expression of multiple copies of the gene (on the plasmid) is completely dependent on the expression of T7 RNA polymerase, which can be easily suppressed because it exists as a single copy.

  There are two examples of vectors with suitable inducible promoters. Others are known to those skilled in the art. The improved nuclease of the present invention is not limited by the selection of the expression system.

  For ligation to the pTTQ18 vector, the DNA of the PCR product containing the Taq polymerase coding region (called mutTaq for the following reasons; SEQ ID NO: 238) was digested with EcoRI and BglII, and standard “sticky end” conditions ( Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1.63-1.69 [1989]) ligated this fragment to the EcoRI / BamHI site of plasmid vector pTTQl8. Expression of this construct replaces the first two residues (Met-Arg) of the native protein with three residues from the vector (Met-Asn-Ser), but changes in the rest of the protein sequence of the PCR product A translational fusion product without (SEQ ID NO: 239) is obtained. Escherichia coli JM109 strain was transformed with the construct, and the transformant was seeded on a plate under incomplete suppression conditions that suppressed the growth of bacteria expressing the natural protein. Such plating conditions can isolate genes that contain naturally occurring mutations, such as those caused by Taq polymerase inaccuracies in the amplification process.

Using this amplification / selection protocol, a clone containing a mutated Taq polymerase gene (mutTaq) was isolated. First, mutants were detected by traits. The temperature stable 5 'nuclease activity in the crude cell extract was normal, but there was little polymerization activity (approximately less than 1% of wild-type Taq polymerase activity). Polymerase activity was determined by primer extension reaction. The reaction was performed in 10 μl of buffer containing 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl. In each reaction, 40 ng of enzyme was used for 10 μM (dT) at 60 ° C. for 30 minutes in the presence of either 10 μM poly (A) ₂₈₆ or 1 μM poly (dA) ₂₇₃ template, 45 μM dTTP and 5 μM Fl-dUTP. ) Extension was performed from _25-30 primer. The reaction was stopped with 10 μl of stop solution (95% formamide, 10 mM EDTA, 0.02% methyl violet dye). Sample (3μl) was fractionated on a 15% acrylamide-denaturing gel (cross-linked 19: 1), and the fraction incorporating Fl-dUTP was quantified using an FMBIO-100 fluorescent gel scanner (Hitachi) equipped with a 505 nm emission filter. did.

  According to DNA sequence analysis of the recombinant gene, two amino acid substitutions (change from A to G at nucleotide position 1394 changes Glu at amino acid position 465 to Gly (numbered according to the natural nucleic acid and amino acid sequence). And another A to G change at nucleotide position 2260 changed Gln at amino acid position 754 to Arg), resulting in a mutation in the polymerase domain. The Gln to Gly mutation is in a non-conservative position, and the Glu to Arg mutation changes the amino acid conserved in virtually all known type A polymerases, so the latter mutation is This is likely to be one of the causes of lowering the synthetic activity. The nucleotide sequence of the construct is set forth in SEQ ID NO: 39. The enzyme encoded by this sequence is expressed as Taq A / G.

得られたPCR産物を、制限エンドヌクレアーゼEcoRIおよびSalIで消化し、EcoRI／SalI消化したプラスミド ベクターpTrc99G（実施例2C1に記載する）に挿入することによってプラスミド pTrcTth-1を作製した。このTth ポリメラーゼ構築物は、5'オリゴヌクレオチドが偶然欠落して1残基のヌクレオチドを欠いている。その結果、ポリメラーゼ遺伝子は読み枠からはずれた。以下のオリゴヌクレオチドを用いて実施例4および5に記載の方法に従い、pTrcTth-1に部位特異的変異導入を行い、この間違いを修正した。

これにより、プラスミド pTrcTth-2を作製した。TthPolのタンパク質および該タンパク質をコードする核酸配列は、それぞれ配列番号：243および244に示した。 b. Initial isolation of TthPol Production of a DNA polymerase enzyme from the bacterial species Thermus thermophilus (Tth) was performed by cloning the gene for this protein into an expression vector and overexpressing it in E. coli cells. Genomic DNA was prepared from 1 vial of ATCC (ATCC # 27634) dried Thermus thermophilus strain HB-8. The DNA polymerase gene was amplified by PCR using the following primers.

The resulting PCR product was digested with the restriction endonucleases EcoRI and SalI and inserted into the EcoRI / SalI digested plasmid vector pTrc99G (described in Example 2C1) to create plasmid pTrcTth-1. This Tth polymerase construct is missing one nucleotide because of the accidental loss of the 5 'oligonucleotide. As a result, the polymerase gene was out of the reading frame. This mistake was corrected by site-directed mutagenesis into pTrcTth-1 according to the method described in Examples 4 and 5 using the following oligonucleotides.

This produced plasmid pTrcTth-2. The TthPol protein and the nucleic acid sequence encoding the protein are shown in SEQ ID NOs: 243 and 244, respectively.

c. Large-scale preparation of recombinant proteins
The recombinant protein was purified as follows using the following technique based on the preparation protocol of Taq DNA polymerase (Engeike et al., Anal. Biochem., 191: 396 [1990]). E. coli cells (JM109 strain) containing either pTrc99A TaqPol or pTrc99GTthPol were inoculated into 3 ml LB containing 100 mg / ml ampicillin and cultured at 37 ° C. for 16 hours. All those cultured overnight was inoculated into LB of 200ml or 350ml containing 100 mg / ml ampicillin, and incubated with vigorous shaking at 37 ° C., the IPTG (1M stock solution) where A ₆₀₀ reached 0.8 Added at a final concentration of 1 mM. Incubation was continued at 37 ° C. for 16 hours.

The induced cells were pelleted and the cell pellet was weighed. An equal volume of 2x DG buffer (100 mM Tris-HCl, pH 7.6, 0.1 mM EDTA) was added and shaken to suspend the pellet. 50 mg / ml lysozyme (Sigma) was added at a final concentration of 1 mg / ml and the cells were incubated for 15 minutes at room temperature. While vortexing, deoxycholic acid (10% solution) was added dropwise to a final concentration of 0.2%. One volume of H ₂ O and one volume of 2 × DG buffer was added and the mixture was sonicated for 2 minutes on ice to reduce the viscosity of the resulting mixture. After sonication, 3M (NH ₄ ) ₂ SO ₄ was added at a final concentration of 0.2 M and the lysate was centrifuged at 14,000 × g for 20 minutes at 4 ° C. The supernatant was taken and incubated at 70 ° C. for 60 minutes. At this point, 10% polyethyleneimine (PEI) was added to 0.25%. After incubating on ice for 30 minutes, the mixture was centrifuged at 14,000 xg for 20 minutes at 4 ° C. At this point, the supernatant was removed and (NH ₄ ) ₂ SO ₄ was added to precipitate the protein as follows.

Two volumes of 3M (NH ₄ ) ₂ SO ₄ were added to precipitate the protein. The mixture was incubated at room temperature overnight for 16 hours and centrifuged at 14,000 xg for 20 minutes at 4 ° C. The protein pellet was suspended in 0.5 ml Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1% Tween 20). For the preparation of Mja FEN-1, solid (NH ₄ ) ₂ SO ₄ was added at a final concentration of 3M (˜75% saturation). The mixture was incubated on ice for 30 minutes and the protein was spun down and suspended as described above.

The suspended protein preparation was quantified by examining the A _279, dialyzed to 50% glycerol containing 100 μg / ml BSA, 20mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% Tween 20,% Nonidet P Saved at -40.

B. Thermus filiformis and Thermus scottodactus DNA polymerase
1. Cloning of Thermus filiformis and Thermus scottodactus
Add 1 ml of Castenholz medium (DSMZ medium 86) to 1 vial of Thermus filiformis (Tfi) freeze-dried product obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellculturen, Braunschweig, Germany, strain # 4687) Inoculated into 500 ml of Castenholz medium that had been warmed. The culture was incubated for 48 hours at 70 ° C. with vigorous shaking. After culturing, the cells were collected by centrifugation at 8000 × g for 10 minutes. The cell pellet was suspended in 10 ml TE (10 mM Tris-HCL, pH 8.0, 1 mM EDTA), 1 ml was removed, and the cells were frozen at −20 ° C. 1 ml was thawed, lysozyme was added to 1 mg / ml, and the cells were incubated at 23 ° C. for 30 minutes. A 20% SDS solution (sodium dodecyl sulfate) was added to a final concentration of 0.5% and then extracted with buffered phenol. The aqueous phase was further extracted with 1: 1 phenol-chloroform and finally with chloroform. One-tenth volume of 3M sodium acetate (pH 5.0) and 2.5 volumes of ethanol were added to the aqueous phase and mixed. DNA was pelleted by centrifugation at 20,000 xg for 5 minutes. The DNA pellet was washed with 70% ethanol and air dried. This was resuspended in 200 μl TE and used directly for amplification. Thermus scotdactus (Tsc, ATCC # 51532) was cultured and genomic DNA was prepared by the method described above for Thermus filiformis.

反応によるPCR産物の長さは約1200塩基対であった。制限酵素NotIおよびSalIで切断し、得られたDNAをNotIおよびSalIで切断したpTrc99aに連結し、pTrc99a-Tfi3'を作製した。遺伝子の5'側の半分を上記の方法で以下に記載の2つのプライマー用いて増幅した。

得られた1300塩基対の断片を制限酵素EcoRIおよびNotIで切断し、EcoRIおよびNotIで切断したpTrc99a-Tfi3'に連結して、pTrc99a-TfiPol（配列番号：249（対応するアミノ酸配列を配列番号：250に示した））を作製した。 The Tfi DNA polymerase I gene (GenBank accession number AF030320) could not be amplified as a single fragment. Therefore, two different fragments were cloned into the expression vector pTrc99a. The two fragments overlap and share the NotI site created by introducing a silent mutation at position 1308 of the open reading frame (ORF) of Tfi DNA polymerase in the PCR oligonucleotide. Using the Advantage cDNA PCR kit (Clonetech) and the following oligonucleotides, the 3 ′ half of this gene was amplified.

The length of the PCR product from the reaction was about 1200 base pairs. Cleaved with restriction enzymes NotI and SalI, and the resulting DNA was ligated to pTrc99a cleaved with NotI and SalI to prepare pTrc99a-Tfi3 ′. The 5 ′ half of the gene was amplified by the method described above using the two primers described below.

The obtained 1300 base pair fragment was cleaved with restriction enzymes EcoRI and NotI, ligated to pTrc99a-Tfi3 ′ cleaved with EcoRI and NotI, and pTrc99a-TfiPol (SEQ ID NO: 249 (corresponding amino acid sequence SEQ ID NO: 250) was produced.

PCR産物を制限酵素EcoRIおよびSalIで切断し、EcoRIおよびSalIで切断したpTrc99aに連結し、pTrc99a-TscPol（配列番号：253（対応するアミノ酸配列を配列番号：254に示した））を作製した。 Using the Advantage cDNA PCR kit (Clonetech) and the following two primers, the DNA polymerase I gene of Thermus scottodactus was amplified.

The PCR product was cleaved with restriction enzymes EcoRI and SalI and ligated to pTrc99a cleaved with EcoRI and SalI to prepare pTrc99a-TscPol (SEQ ID NO: 253 (corresponding amino acid sequence is shown in SEQ ID NO: 254)).

2. Expression and purification of Thermus filiformis and Thermus scotdactus For protein expression, plasmids were transformed into protease-deficient E. coli strain BL21 (Novagen) or strain JM109 (Promega Corp., Madison, Wis.). Flasks containing LB containing 100 μg / ml ampicillin were inoculated with single colonies of LB plates or frozen stocks of appropriate strains. After culturing at 37 ° C. for several hours with vigorous shaking, induction was performed by adding 200 μl of 1M isopropyl galactoside (IPTG) to the culture solution. The culture was continued at 37 ° C. for 16 hours, and then collected. After centrifuging at 8000 × g for 15 minutes to pellet the cells, the cell pellet was suspended in 5 ml of TEN (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl). 100 μl of 50 mg / ml lysozyme was added and the cells were incubated for 15 minutes at room temperature. Deoxycholic acid (10%) was added at a final concentration of 0.2%. After mixing well, the cell lysate was sonicated on ice for 2 minutes to reduce the viscosity of the mixture. Cell debris was pelleted by centrifugation at 20,000 xg for 15 minutes at 4 ° C. The supernatant was removed, 10% polyethylimine (PEI) was added at 0.25%, and the mixture was kept at 70 ° C. for 30 minutes. After incubation on ice for 30 minutes, the mixture was centrifuged at 20,000 xg for 20 minutes at 4 ° C. At this point, the supernatant containing the enzyme was taken, 1.2 g of ammonium sulfate was added, and the mixture was incubated at 4 ° C. for 1 hour to precipitate the protein. Proteins were pelleted by centrifugation at 20,000 xg for 10 minutes at 4 ° C. The pellet was resuspended in 4 ml of HPLC buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA). The protein was further purified by affinity chromatography using an Econo-Pac heparin cartridge (BioRad) and a Dionex DX 500 HPLC instrument. Briefly, the cartridge was equilibrated with HPLC buffer A, the enzyme extract was added to the column and eluted with a linear gradient of NaCl (0-2M) in the same buffer. Pure protein elutes between 0.5 and 1M NaCl. The enzyme peak was collected and dialyzed against 50% glycerol, 20 mM Tris-HC1, pH 8, 50 mM KCl, 0.5% Tween 20, 0.5% nonidet P40, 100 mg / ml BSA.

C. Preparation of polymerase mutants with reduced polymerase activity and invariant 5 ′ nuclease activity All mutants prepared in Section C were expressed and purified by the method described in Example 2A1C.

1. Modified TaqPol gene: TaqDN
A Taq DNA polymerase mutant deficient in polymerization activity called TaqDN was constructed. TaqDN nuclease contains an asparagine residue in place of the wild-type aspartate residue 785 (D785N).

  From the gene encoding Taq A / G, DNA encoding TaqDN nuclease was constructed by two site-directed mutagenesis methods. First, wild-type TaqPol gene was prepared by changing G at position 1397 and G at position 2264 of the Taq A / G gene (SEQ ID NO: 238) to A, respectively. In the second round of mutagenesis, the wild-type TaqPol gene was converted to the Taq DN gene by changing G at position 2356 to A. These operations were performed as follows.

  Taq A / G nuclease-encoding DNA was recloned from the pTTQ18 plasmid into the pTrc99A plasmid (Pharmacia) by a two-step method. First, the pTrc99A cloning vector was prepared by modifying the pTrc99A vector by removing G at position 270 in the pTrc99A map. At this stage, the pTrc99A plasmid DNA was cleaved with NcoI, and the 3 ′ recessed end was blunted using Klenow fragment of E. coli polymerase I for 15 minutes at 37 ° C. in the presence of 4 types of dNTPs. The Klenow fragment was inactivated by incubation at 65 ° C. for 10 minutes, and the plasmid DNA was cleaved with EcoRI. Again, the ends were blunted with Klenow fragment in the presence of 4 types of dNTPs at 37 ° C. for 15 minutes. Next, the Klenow fragment was inactivated by incubating at 65 ° C. for 10 minutes. Plasmid DNA was ethanol precipitated and recircularized by ligation. This was used to transform E. coli JM109 cells (Promega). Plasmid DNA was isolated from a single colony and the deletion of the G at position 270 in the pTrc99A map was confirmed by DNA sequencing.

  In the second step, the DNA encoding Taq A / G nuclease is removed from the pTTQ18 plasmid using EcoRI and SalI, the DNA fragment containing the Taq A / G nuclease gene is separated on a 1% agarose gel, and Geneclean II Isolated with kit (Bio 101, Vista, CA). The purified fragment was ligated into the pTrc99G vector cut with EcoRI and SalI. The ligation mixture was used to transform E. coli JM109 competent cells (Promega). Plasmid DNA was isolated from a single colony and the Taq A / G nuclease gene insert was confirmed by restriction enzyme analysis using EcoRI and SalI.

  Plasmid DNA containing the pTrcAG Taq A / G nuclease gene was cloned into the pTrc99A vector, and the overnight culture was purified from 200 ml JM109 using the QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, CA) according to the manufacturer's protocol. Two types of mutagenesis primers E465 (SEQ ID NO: 255) (Integrated DNA Technologies, Iowa) and R754Q (SEQ ID NO: 256) (Integrated DNA Technologies, Iowa), and the selection primer trans-oligonucleotide AlwNI / SpeI ( Using Clontech, Palo Alto, CA, Cat. No. 6488-1), following the protocol of TRANSFORMER site-directed mutagenesis kit (Clontech, Palo Alto, CA) A type TaqPol gene (pTrcWT) was prepared.

  The pTrcWT plasmid DNA containing the wild-type TaqPol gene was cloned into the pTrc99A vector, and the overnight culture was purified from 200 ml of JM109 using the QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, CA) according to the manufacturer's protocol. Next, using the mutagenesis primer D785N (SEQ ID NO: 257) (Integrated DNA Technologies) and the selection primer switch oligonucleotide SpeI / AlwNI (Clontech, Palo Alto, CA, Catalog No. 6373-1), TRANSFORMER According to the protocol of the site-directed mutagenesis kit (Clontech, Palo Alto, CA), mutation was induced in pTrcWT to prepare a plasmid containing DNA encoding Taq DN nuclease. The DNA encoding Taq DN nuclease is shown in SEQ ID NO: 258, and the amino acid sequence of Taq DN nuclease is shown in SEQ ID NO: 259.

これにより、プラスミド pTrcTthDNを作製した。変異体タンパク質、およびタンパク質をコードする核酸配列は、それぞれTthDN 配列番号：261および262とした。 2. Modified TthPol gene: Tth DN
Tth DN constructs were generated by inducing mutations in the above TthPol. By the site-directed mutagenesis described above, the sequence encoding aspartic acid at position 787 was changed to a sequence encoding asparagine. Mutation of pTrcTth-2 was performed using the following oligonucleotides:

Thereby, plasmid pTrcTthDN was prepared. The mutant protein and the nucleic acid sequence encoding the protein were TthDN SEQ ID NOs: 261 and 262, respectively.

配列136-037-05である。
いずれの変異誘発反応にも、選択プライマーであるトランスオリゴ AlwNI／SpeI（Clontech, カタログ番号6488-1）を用いた。得られた変異体遺伝子をTaq DN HT（配列番号：265、核酸配列；配列番号：266、 アミノ酸配列）およびTth DN HT（配列番号：267、核酸配列；配列番号：268、アミノ酸配列）と名付けた。 3. Taq DN HT and Tth DN HT
A tag containing a 6-residue amino acid histidine (His-tag) was added to the carboxyl terminus of Taq DN and Tth DN. Site-directed mutagenesis was performed using the TRANSFORMER site / site-directed mutagenesis kit (Clontech) according to the manufacturer's instructions. The mutation oligonucleotides used for plasmids pTaq DN and pTth DN are:
Array 117-067-03,

Arrangement 136-037-05.
In each mutagenesis reaction, the selection primer trans-oligo AlwNI / SpeI (Clontech, catalog number 6488-1) was used. The obtained mutant genes were named Taq DN HT (SEQ ID NO: 265, nucleic acid sequence; SEQ ID NO: 266, amino acid sequence) and Tth DN HT (SEQ ID NO: 267, nucleic acid sequence; SEQ ID NO: 268, amino acid sequence). It was.

4. Purification of Taq DN HT and Tth DN HT Taq DN HT and Tth DN HT proteins were expressed in E. coli strain JM109 as in Example 2B2. After ammonium sulfate precipitation and centrifugation, the protein pellet was suspended in 0.5 ml of Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.1% Tween 20). The protein was further purified by affinity chromatography using His-binding resin and buffer kit (Novagen) according to the manufacturer's instructions. 1 ml of His-binding resin was transferred to the column and washed with sterile water 3 times the column volume. 5 volumes of 1 × charge buffer was loaded and equilibrated with 3 volumes of 1 × binding buffer. 4 ml of 1 × binding buffer was added to the protein sample and the sample solution was added to the column. After washing with 3 ml of 1 × binding buffer and 3 ml of 1 × wash buffer, the bound his-tag protein was eluted with 1 ml of 1 × elution buffer. The pure enzyme was then dialyzed against 50% glycerol, 20 mM Tris-HC1, pH 8.0, 50 mM KCl, 0.5% Tween 20, 0.5% nonidet P40, 100 μg / ml BSA. The absorbance at 279 nm was measured to determine the enzyme concentration.

Example 3
By transferring the N-terminal part of the DNA polymerase domain, TaqPol can confer the RNA-dependent 5 'nuclease activity of TthPol.
A. Preparation and purification of substrate structure with either DNA or RNA target strand
By means of standard phosphoramidite chemistry (Glen Research) using PerSeptive Biosystems equipment, downstream probes (SEQ ID NO: 162) and upstream probes (SEQ ID NO: 161), and IL-6 DNA (SEQ ID NO: 163) (FIG. 10). ) The target strand was synthesized. A synthetic RNA-DNA chimeric IrT target (FIG. 20A) labeled with biotin at the 5 ′ end was synthesized using 2′-ACE RNA chemistry (Dharmacon Research). The 2'-protecting group was removed by acid-catalyzed hydrolysis according to the manufacturer's instructions. The downstream probe labeled at the 5 ′ end with 5′-fluorescein (F1) or 5′-tetrachloro-fluorescein (TET) was purified by reverse phase HPLC using a Resource Q column (Amersham-Phannacia Biotech). A cloned fragment of human IL-6 cDNA (sequence of nucleotides 64 to 691 as described in May et al., Proc. Natl. Acad. Sci., 83: 8957 [1986]) was isolated using T7 using the Megascript kit (Ambion). A 648 nucleotide IL-6 RNA target (SEQ ID NO: 160) (FIG. 10) was synthesized by run-off transcription with RNA polymerase. Finally, after separation by 20% denaturing polyacrylamide gel, the main band was cut out and eluted to purify the whole oligonucleotide. The absorbance at 260 nm was measured and the oligonucleotide concentration was determined. Biotinylated IrT target in buffer containing 10 mM MOPS, pH 7.5, 0.05% Tween20, 0.05% NP-40 and 10 μg / ml tRNA was incubated for 10 minutes at room temperature with a 5-fold excess of streptavidin (Promega) .

B. Introduction of restriction enzyme sites for production of chimeras The restriction enzyme sites used for the production of the following chimeric proteins were selected for convenience. The restriction enzyme sites in the following examples were designed to be arranged in the surrounding region (see FIGS. 6, 7 and 19) shown by the crystal structure of the functional domain and other analyses. Similar constructs can be made for type A polymerase genes from other related organisms using different sites created by natural or site-directed mutagenesis. It is preferred that any mutation in terms of protein function is silent. By examining the nucleic acid sequence and the amino acid sequence of the protein, mutations that do not affect the amino acid sequence corresponding to the nucleic acid sequence can be introduced. If the desired nucleic acid mutation affects the amino acid, it can be altered to have the same or similar properties as the replacement of the new amino acid. If such an option is not possible, it can be examined whether changes in the nucleic acid resulted in changes in the activity, specificity or function of the protein by testing the function of the mutant enzyme. The present invention is not limited by the particular restriction enzyme site selected or introduced to produce the improved enzyme of the present invention.

C. Generation of Tth DN RX HT and Taq DN RX HT Mutagenesis was performed, and unique restriction enzyme sites were additionally introduced at three locations in the polymerase domain of both Taq DN HT and Tth DN HT enzymes. . Site-directed mutagenesis was performed using Clonetech's Transformer site-directed mutagenesis kit according to the manufacturer's instructions. For all mutagenesis reactions described, one of two different selection primers, trans-oligo AlwNI / SpeI or switch oligo SpeI / AlwNI (Clontech, Palo Alto CA catalog number 6488-1 or catalog number 6373-1) Either one was used. The selection oligo utilized for a given reaction depends on the choice of restriction enzyme sites in the vector. All mutagenesis primers were synthesized using standard synthetic chemistry. The resulting colony was expressed in E. coli strain JM109.

を用いて、NotI部位（アミノ酸の328位）を生成させた。センス鎖変異プライマー

を用いて、BstI（アミノ酸382位）およびNdeI（アミノ酸443位）部位を両方の遺伝子に導入した。変異体プラスミドを過剰発現し、Qiagen QiaPrep Spin Mini Prepキット（カタログ番号27106）を用いて精製した。DNAシーケンシングおよび制限酵素マッピングを用いて、制限酵素部位の存在に関してベクターを試験した。これらの構築物をTth DN RX HT（DNA配列、配列番号：273；アミノ酸配列、配列番号：274）およびTaq DN RX HT（DNA配列、配列番号：275；アミノ酸配列、配列番号：276）と名付けた。 Mutagenesis primers corresponding to the sense strands of Taq DN HT and Tth DN HT genes, respectively

Was used to generate a NotI site (amino acid position 328). Sense strand mutation primer

Was used to introduce BstI (amino acid position 382) and NdeI (amino acid position 443) sites into both genes. The mutant plasmid was overexpressed and purified using the Qiagen QiaPrep Spin Mini Prep kit (Catalog No. 27106). The vector was tested for the presence of restriction enzyme sites using DNA sequencing and restriction enzyme mapping. These constructs were named Tth DN RX HT (DNA sequence, SEQ ID NO: 273; amino acid sequence, SEQ ID NO: 274) and Taq DN RX HT (DNA sequence, SEQ ID NO: 275; amino acid sequence, SEQ ID NO: 276). .

D. Chimera Restriction endonuclease sites EcoRI (E) and BamHI (B) common to both genes; Cloning vector site SalI (S); and a novel site introduced at the homologous position of both genes by site-directed mutagenesis A chimeric construct shown in FIG. 19 was prepared by exchanging homologous DNA fragments obtained with NotI (N), BstBI (Bs), and NdeI (D). In making these chimeric enzymes, two different DNA fragments are ligated together to obtain the final construct. A larger DNA fragment containing a plasmid vector and part of the Taq or Tth (or part of both) sequence is called a “vector”. A smaller DNA fragment containing the sequence of Taq or Tth (or part of both) polymerase is called an “insert”.

  All restriction enzymes were purchased from New England Biolabs or Promega. The reaction was performed using the attached buffer according to the manufacturer's instructions. Approximately 500 ng of DNA per reaction was reacted in a volume of 20 μl at the optimum temperature for each enzyme. Two or more enzymes were used simultaneously in a single reaction (double digest) if the enzymes were compatible with respect to reaction buffer conditions and reaction temperature. If the enzyme may be incompatible with buffer conditions, the enzyme that required the lowest salt conditions was used first. After the reaction was completed, the second reaction was carried out with the buffer conditions optimal or better for the second enzyme. This is a commonly used restriction enzyme digestion procedure known in the art of basic molecular biology (Maniatis, supra).

  For optimum ligation efficiency, the restriction enzyme digested fragment was isolated on a gel. 2 μl of 10 × loading dye (50% glycerol, 1 × TAE, 0.5% bromophenol blue) was added to 20 μl reaction. All volumes were added and run on a 1% 1 × TAB agarose gel containing 1 μl of 1% ethidium bromide solution per 100 ml of agarose gel solution. The digested fragments were visualized under UV light and excised from the appropriate fragment (determined by size) gel. These fragments were then purified using the Qiagen Gel Extraction kit (Cat # 28706) according to the manufacturer's instructions.

  Ligation was performed in a volume of 10 μl using 400 units per reaction of T4 DNA ligase enzyme from New England Biolabs (Cat. No. 202L) and the attached reaction buffer. For each Qiagen purified fragment (each DNA is approximately 20-50 ng, depending on recovery from isolation by gel), a ligation reaction was performed with 1 μl for 1 hour at room temperature. Next, E. coli strain JM109 was transformed with the ligation product and inoculated into an appropriate growth / selection medium such as LB containing 100 μg / ml ampicillin for selection of transformants.

  For each ligation reaction, six transformants were examined for the presence or absence of the desired construct. Plasmid DNA was purified and isolated using the QiaPrep Spin Mini Prep kit according to the manufacturer's instructions. The construct was confirmed using DNA sequencing and restriction enzyme mapping.

  The expression and purification of the chimeric enzyme was performed as follows. The plasmid was transformed into E. coli strain JM109 (Promega). A logarithmic growth phase culture of JM109 (200 ml) was induced with 0.5 mM IPTG (Promega), further cultured for 16 hours, and then collected. The pelleted cells were lysed by incubating in 5 ml of 10 mM Tris-HCl, pH 8.3, 1 mM EDTA, 0.5 mg / ml lysozyme for 15 minutes at room temperature to prepare a crude extract containing soluble protein. The lysate was mixed with 5 ml of 10 mM Tris-HCl, pH 7.8, 50 mM KCl, 1 mM EDTA, 0.5% Tween 20, 0.5% Nonidet P-40 and heated at 72 ° C. for 30 minutes. Cell debris was removed by centrifugation at 12,000 xg for 5 minutes. Final protein purification was performed by affinity chromatography using an Econo-Pac heparin cartridge (Bio-Rad) and a Dionex DX 500 HPLC instrument. Briefly, the cartridge was equilibrated with 50 mM Tris-HCl, pH 8, 1 mM EDTA, and the enzyme extract was dialyzed against the same buffer. This was added to the column and eluted with a linear gradient of NaCl (0-2M) in the same buffer. The HPLC purified protein was dialyzed and stored in 50% (vol / vol) glycerol, 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5% Tween 20, 0.5% Nonidet P-40 and 100 μg / ml BSA. The enzyme was purified by SDS-PAGE until uniform, and the enzyme concentration was determined by measuring absorbance at 279 nm.

1. Construction of TaqTth (N) and TthTaq (N) Modifications involving the polymerase domains of these two enzymes were first performed. The nuclease domain (N-terminus of the protein) was separated from the polymerase domain (C-terminal portion of the protein) by cleaving the respective genes using the restriction endonucleases EcoRI and NotI. A fragment of about 900 base pairs derived from the Tth DN RX HT gene was cloned into the homologous site of the Taq DN RX HT gene. In addition, a fragment of about 900 base pairs derived from the Taq DN RX HT gene was cloned into the homologous site of the Tth DN RX HT gene. This includes a Taq DN RX HT 5 'nuclease domain and a Tth DN RX HT polymerase domain (N) (DNA sequence, SEQ ID NO: 69; amino acid sequence, SEQ ID NO: 2) and Tth DN RX HT 5' nuclease Two types of chimeras were obtained: TthTaq (N) (DNA sequence, SEQ ID NO: 70; amino acid sequence, SEQ ID NO: 3) containing the domain and Taq DN RX HT polymerase domain.

2. Construction of TaqTth (NB)
The Taq DN RX HT construct was cut with the enzymes NdeI and BamHI and the larger vector fragment was isolated by gel as described above. In addition, the Tth DN RX HT construct was cleaved with NdeI and BamHI, and a smaller (about 795 base pair) Tth fragment was isolated and purified on a gel. The Tth NdeI-BamHI insert was ligated to the Taq NdeI-BamHI vector as described above to produce TaqTth (NB) (DNA sequence, SEQ ID NO: 71; amino acid sequence, SEQ ID NO: 4).

3. Construction of TaqTth (BS)
The Taq DN RX HT construct was cut with the enzymes BamHI and SalI and the larger vector fragment was isolated by gel in the manner described above. In addition, the Tth DN RX HT construct was cleaved with BamHI and SalI, and a smaller (about 741 base pair) Tth fragment was isolated and purified on a gel. The Tth BamHI-SalI insert was ligated to the Taq BamHI-SalI vector by the method described above to produce TaqTth (BS) (DNA sequence, SEQ ID NO: 72; amino acid sequence, SEQ ID NO: 5).

4. Construction of TaqTth (ND)
The Taq DN RX HT construct was cut with the enzymes NotI and NdeI and the larger vector fragment was isolated as described above. In addition, the Tth DN RX HT construct was cleaved with NotI and NdeI, and a smaller (about 345 base pair) Tth fragment was isolated and purified on a gel. The Tth NotI-NdeI insert was ligated to the Taq NotI-NdeI vector by the method described above to produce TaqTth (ND) (DNA sequence, SEQ ID NO: 73; amino acid sequence, SEQ ID NO: 6).

5. Construction of TaqTth (DB)
The Taq DN RX HT construct was cut with the enzymes NdeI and BamHI, and the larger vector fragment was isolated as described above. In addition, the Tth DN RX HT constructs NdeI and BamHI were cleaved, and a smaller (about 450 base pair) Tth fragment was isolated and purified on a gel. The Tth NdeI-BamHI insert was ligated to the Taq NdeI-BamHI vector as described above to produce TaqTth (DB) (DNA sequence, SEQ ID NO: 74; amino acid sequence, SEQ ID NO: 7).

6. Construction of TaqTth (Bs-B)
The Taq DN RX HT construct was cut with the enzymes BstBI and BamHI and the larger vector fragment was isolated as described above. In addition, the Tth DN RX HT construct was cleaved with BstBI and BamHI, and a smaller (about 633 base pair) Tth fragment was isolated and purified on a gel. The Tth NdeI-BamHI insert was ligated to the Taq NdeI-BamHI vector as described above to produce TaqTth (Bs-B) (DNA sequence, SEQ ID NO: 75; amino acid sequence, SEQ ID NO: 8).

7. Construction of TaqTth (N-Bs)
The Taq DN RX HT construct was cut with the enzymes NotI and BstBI and the larger vector fragment was isolated as described above. In addition, the Tth DN RX HT construct was cleaved with NotI and BstBI, and a smaller (about 162 base pair) Tth fragment was isolated and purified on a gel. The Tth NotI-BstBI insert was ligated to the Taq NotI-BstBI vector as described above to produce TaqTth (N-Bs) (DNA sequence, SEQ ID NO: 76; amino acid sequence, SEQ ID NO: 9).

8. Construction of TthTaq (BS)
The Tth DN RX HT construct was cut with the enzymes BamHI and SalI and the larger vector fragment was isolated as described above. In addition, the Taq DN RX HT construct was cleaved with BamHI and SalI, and a smaller (about 741 base pair) Tth fragment was isolated and purified on a gel. The Taq BamHI-SalI insert was ligated to the Tth BamHI-SalI vector to produce TthTaq (BS) (DNA sequence, SEQ ID NO: 77; amino acid sequence, SEQ ID NO: 10).

9. Construction of Tth Taq (NB)
The Tth DN RX HT construct was cut with the enzymes NotI and BamHI, and the larger vector fragment was isolated as described above. In addition, the Taq DN RX HT construct was cleaved with NotI and BamHI, and a smaller (about 795 base pair) Tth fragment was isolated and purified on a gel. The Taq NotI-BamHI insert was ligated to the Tth NotI-BamHI vector to produce TthTaq (NB) (DNA sequence, SEQ ID NO: 78; amino acid sequence, SEQ ID NO: 11).

  Characterization of the cleavage activity of these chimeric proteins was performed by the method described in Example 1 (Section A) and a comparison of the cleavage cycling rates for RNA targets is shown in FIG. As further explained in the description of the invention, these data are found in the middle third of the TthPol protein in conferring a TthPol-like RNA-dependent cleavage activity on a chimeric protein containing a portion of TaqPol. Indicates that the element is important.

Example 4
Modifications that affect RNA-dependent 5 'nuclease activity do not necessarily affect RNA-dependent DNA polymerase activity
TthPol is known to be a more active RNA template-dependent DNA polymerase than TaqPol (Myers and Gelfand, Biochemistry 30: 7661 [1991]). To investigate whether the RNA template-dependent 5 'nuclease activity of the Thermus DNA Pol I enzyme is involved in RNA-dependent polymerase activity, the D785N and D787N mutations used for the construction of polymerase-deficient TaqPol and TthPol, respectively, Reversed. TaqTth (N) (DNA sequence, SEQ ID NO: 79; amino acid sequence, SEQ ID NO: 12), TaqTth (NB) (DNA sequence, SEQ ID NO: 80; amino acid sequence, SEQ ID NO: 13), TaqTth (BS) (DNA Sequence, SEQ ID NO: 81; Amino acid sequence, SEQ ID NO: 14) Chimera and TaqPol (W417L / G418K / E507Q) (DNA sequence, SEQ ID NO: 82; Amino acid sequence, SEQ ID NO: 15) Mutant proteins also have polymerase activity Recovered.

  By inserting a natural non-DN sequence BamHI to SalI fragment into the selected chimeric or mutant enzyme, all of the enzyme mutants recovered the polymerase function. For example, the mutant construct TaqTth (N-B) is cleaved with the restriction enzyme BamHI (about 593 of the amino acid) and the restriction enzyme SalI (about 840 of the amino acid). Larger vector fragments were purified by gel in the manner of Example 3D. The native TaqPol construct was also cleaved with the restriction endonucleases BamHI and SalI, and a smaller insert containing the natural amino acid sequence was purified by gel. Next, the inserted fragment was ligated to the vector detailed in Experimental Example 3D.

The polymerase activity of these proteins was assessed by extending dT _25-35 oligonucleotide primers with fluorescein labeled dUTP in the presence of poly (dA) or poly (A) template. The primer extension reaction was performed in 10 μl buffer containing 10 mM MOPS, pH 7.5, 5 mM MgSO ₄ , 100 mM KCl. Extend 10 μM (dT) _25-30 primer for 30 minutes at 60 ° C. with 40 ng enzyme in the presence of 10 μM poly (A) ₂₈₆ or 1 μM poly (dA) ₂₇₃ template and 45 μM dTTP and 5 μM Fl-dUTP I let you. The reaction was stopped using 10 μl stop solution (95% formamide, 10 mM EDTA, 0.02% methyl violet dye). The sample (3 μl) was fractionated with a 15% acrylamide denaturing gel, and the fraction incorporating Fl-dUTP was quantified using the FMBIO-100 fluorescent gel scanner (Hitachi) equipped with the above-mentioned 505 nm filter.

  As shown in FIG. 16, the DNA-dependent polymerase activities in all of the constructs used in this experiment were very similar, but the RNA-dependent polymerase activities of TthPol, TaqTth (N) and TaqTth (BS) were TaqPol. , TaqTth (NB) and TaqPol W417L / G418K / E507Q mutant activity at least 6 times higher. From these analysis results, the high RNA-dependent DNA polymerase activity of TthPol is determined by the C-terminal half of the polymerase domain (roughly amino acids 593 to 830), and the RNA-dependent 5 ′ nuclease and polymerase activity are independent of each other. It can be concluded that it is controlled by different areas.

Example 5
Specific point mutants of Taq DN RX HT based on information obtained from the chimera search. According to the chimera search (Example 3, above), the portion of the TthPol sequence that determines the high activity of the RNA-dependent 5 'nuclease. Is suggested to contain a BstBI-BamHI region located approximately between amino acids 382 and 593. When comparing the amino acid sequences in the BstBI and BamHI regions of Tth DN RX HT and Taq DN RX HT (SEQ ID NOs: 165 and 164, respectively), only a difference of 25 residues is found (FIG. 13). Among these, 12 amino acid changes are conservative and 13 residue differences result in charge changes. Analysis of the chimeric enzyme suggests that there are important mutations in both the BstBI-NdeI and NdeI-BamHI regions of Tth DN RX HT. HT specific amino acids were introduced into the BstBI-NdeI and NdeI-BamHI regions of TaqTth (DB) and TaqTth (ND), respectively.

  Six Tth DN RX HT specific substitutions were introduced into the BstBI-NdeI region of TaqTth (D-B) by single or double amino acid mutagenesis. Similarly, 12 kinds of Tth DN RX HT specific amino acid mutations were introduced at the homologous position of the NdeI-BamHI region of TaqTth (N-D).

  To obtain sufficient starting material for any mutagenesis reaction, plasmid DNA was purified from 200 ml JM109 cultured overnight using the QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.) According to the manufacturer's protocol. All site-specific mutations were introduced according to the manufacturer's protocol, Transformer site-directed mutagenesis kit (using Clontech). Specific sequence information of the mutagenesis primer used at each site is shown below. For all mutagenesis reactions described, one of two different selection primers, trans-oligo AlwNI / SpeI or switch-oligo SpeI / AlwNI (Clontech, Palo Alto, CA catalog number 6488-1 or catalog number 6373-1) used. The selection oligo utilized for a given reaction depends on the choice of restriction enzyme sites in the vector. All mutagenesis primers were synthesized using standard synthetic chemistry. Colonies were obtained with E. coli strain JM109.

を用いてpTrc99A TaqTth（D-B）DNAの部位特異的変異誘発を実施し、E404H変異を導入した。 1. Construction of TaqTth (DB) E404H (DNA sequence, SEQ ID NO: 83; amino acid sequence, SEQ ID NO: 16) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99A TaqTth (DB) DNA to introduce the E404H mutation.

を用いてpTrc99A TaqTth（D-B）DNAの部位特異的変異誘発を実施し、F413HおよびA414R変異を導入した。 2. Construction of TaqTth (DB) F413H / A414R (DNA sequence, SEQ ID NO: 84; amino acid sequence, SEQ ID NO: 17)
Primers for DNA mutagenesis:

Was used for site-directed mutagenesis of pTrc99A TaqTth (DB) DNA to introduce F413H and A414R mutations.

を用いてpTrc99A TaqTth（D-B）DNAの部位特異的変異誘発を実施し、W417LおよびG418K変異を導入した。 3. Construction of TaqTth (DB) W417L / G418K (DNA sequence, SEQ ID NO: 85; amino acid sequence, SEQ ID NO: 18) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99A TaqTth (DB) DNA to introduce W417L and G418K mutations.

を用いてpTrc99A TaqTth（ND-B）DNAの部位特異的変異誘発を実施し、A439R変異を導入した。 4. Construction of TaqTth (DB) A439R (DNA sequence, SEQ ID NO: 86; amino acid sequence, SEQ ID NO: 19) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99A TaqTth (ND-B) DNA to introduce the A439R mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、L415変異を導入した。 5. Construction of TaqTth (ND) L451R (DNA sequence, SEQ ID NO: 87; amino acid sequence, SEQ ID NO: 20) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the L415 mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、L415Q変異を導入した。 6. Construction of TaqTth (ND) R457Q (DNA sequence, SEQ ID NO: 88; amino acid sequence, SEQ ID NO: 21) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the L415Q mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、V463L変異を導入した。 7. Construction of TaqTth (ND) V463L (DNA sequence, SEQ ID NO: 89; amino acid sequence, SEQ ID NO: 22) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the V463L mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、A468R変異を導入した。 8. Construction of TaqTth (ND) A468R (DNA sequence, SEQ ID NO: 90; amino acid sequence, SEQ ID NO: 23) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the A468R mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、A472E変異を導入した。 9. Construction of TaqTth (ND) A472E (DNA sequence, SEQ ID NO: 91; amino acid sequence, SEQ ID NO: 24) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the A472E mutation.

を用いてpTrc99AtaqTfh（N-D）DNAの部位特異的変異誘発を実施し、G499R変異を導入した。 10. Construction of TaqTth (ND) G499R (DNA sequence, SEQ ID NO: 92; amino acid sequence, SEQ ID NO: 25) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTfh (ND) DNA to introduce the G499R mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、E507Q変異を導入した。 11. Construction of TaqTth (ND) E507Q (DNA sequence, SEQ ID NO: 93; amino acid sequence, SEQ ID NO: 26) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the E507Q mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、Y535H変異を導入した。 12. Construction of TaqTth (ND) Y535H (DNA sequence, SEQ ID NO: 94; amino acid sequence, SEQ ID NO: 27) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the Y535H mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、S543N変異を導入した。 13. Construction of TaqTth (ND) S543N (DNA sequence, SEQ ID NO: 95; amino acid sequence, SEQ ID NO: 28) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the S543N mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、I546V変異を導入した。 14. Construction of TaqTth (ND) I546V (DNA sequence, SEQ ID NO: 96; amino acid sequence, SEQ ID NO: 29) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce the I546V mutation.

を用いてpTrc99AtaqTth（N-D）DNAの部位特異的変異誘発を実施し、D551SおよびI553V変異を導入した。 15. Construction of TaqTth (ND) D551S / I553V (DNA sequence, SEQ ID NO: 97; amino acid sequence, SEQ ID NO: 30) Primer for mutagenesis:

Was used for site-directed mutagenesis of pTrc99AtaqTth (ND) DNA to introduce D551S and I553V mutations.

16. Construction of TaqDN RX HT W417L / G418K / E507Q (DNA sequence, SEQ ID NO: 98; amino acid sequence, SEQ ID NO: 31)
A triple mutant of TaqDN RX HT W417L / G418K / E507Q was prepared by combining TaqTth (DB) W417L / G418K with TaqTth (ND) E507Q. TaqTth (DB) W417L / G418K was cleaved with restriction enzymes NdeI and BamHI, and a larger vector fragment was isolated by the method described in detail in Example 3. Also, the TaqTth (ND) E507Q construct was cleaved with NdeI and BamHI, and a small (about 795 base pair) fragment was isolated and purified on a gel by the method described in detail in Example 3. Example 3 The NdeI-BamHI insert was ligated to a gel purified vector as detailed above.

を用いて、部位特異的変異誘発反応を実施し、アミノ酸418位のKを、野生型アミノ酸Gに戻した。Transformer部位特異的変異誘発キット（Clonetech）を用いて製造元の説明書に従い、実験例4に記載する方法で部位特異的変異誘発を実施した。 17. Construction of TaqDN RX HT W417L / E507Q (DNA sequence, SEQ ID NO: 99; amino acid sequence, SEQ ID NO: 32) Starting from the above TaqDN RX HT W417L / G418K / E507Q, primers for mutagenesis:

Was used to perform a site-directed mutagenesis reaction, and K at amino acid position 418 was changed back to wild-type amino acid G. Site-directed mutagenesis was performed by the method described in Experimental Example 4 using the Transformer site-directed mutagenesis kit (Clonetech) according to the manufacturer's instructions.

を用いて、部位特異的変異誘発反応を実施し、アミノ酸417位のLを、野生型アミノ酸Wに戻した。Transformer部位特異的変異誘発キット（Clonetech）を用いて製造元の説明書に従い、実験例4に記載する方法で部位特異的変異誘発を実施した。 18. Construction of TaqDN RX HT G418K / E507Q (DNA sequence, SEQ ID NO: 100; amino acid sequence, SEQ ID NO: 33) Starting from the above TaqDN RX HT W417L / G418K / E507Q, primers for mutagenesis:

Was used to perform a site-directed mutagenesis reaction, and L at amino acid position 417 was changed back to wild-type amino acid W. Site-directed mutagenesis was performed by the method described in Experimental Example 4 using the Transformer site-directed mutagenesis kit (Clonetech) according to the manufacturer's instructions.

  Mutant proteins were expressed and purified as described in detail in Example 3, and the cleavage activity of these proteins was characterized by the method described in Example 1A). A comparison of the cleavage cycling rates of selection of these mutant proteins against the RNA target is shown in FIG. As further explained in the “Description of the Invention”, these data indicate that TaqPol's combination with a portion of TthPol that cannot independently enhance RNA-dependent activity (ie, the DB and ND portions of Tth). It shows that the amino acids of regions 417/418 and amino acid 507 are important for conferring TthPol-like RNA-dependent cleavage activity on a chimeric protein containing a region. Taq DN RX HT variants containing only W417L, G418K and E507Q substitutions were made as described in “Description of the Invention”. By comparing the cleavage rate of Tth DN RX HT to IL-6 RNA substrate with the method described in Example 1, it is clear that these mutations sufficiently increase Taq DN RX HT activity to Tfh DN RX HT levels I made it. Figure 15, Taq DN RX HT W417L / G418K / E507Q and Taq DN RX HT G418K / E507Q mutants have 1.4 times higher activity than Tth DN RX HT and 4 times higher activity than Taq DN RX HT, This shows that the Taq DN RX HT W417L / E507Q mutant has 3 times higher enzyme activity than Taq DN RX HT. These results indicate that TthPol's K418 and Q507 are particularly important amino acids that provide higher RNA-dependent 5 'nuclease activity compared to TaqPol.

Example 6
Taq DN RX HT G418K / E507Q 5 'nuclease is similar to Tth DN RX HT in terms of RNA-dependent 5' nuclease in terms of optimal salt and temperature
Assays that differ in temperature, salt concentration, and divalent ions to determine whether the G418K / E507Q mutation resulted in significant property changes in the Taq DN RX HT mutant in addition to increasing the cleavage rate of the RNA target Under conditions, RNA template-dependent 5 'nuclease assay, Taq DN RX HT G418K / E507Q (SEQ ID NO: 33), Taq DN RX HT (SEQ ID NO: 276), and Tth DN RX HT (SEQ ID NO: 274) The enzymes were compared. As shown in FIG. 20A, the upstream DNA and substrate template RNA strands used in this study were bound to a single IrT molecule (SEQ ID NO: 166). The labeled downstream probe (SEQ ID NO: 167) was in excess. The 5 'end of the target RNA strand was blocked with a biotin-streptavidin complex to prevent nonspecific degradation by the enzyme during the reaction (Lyamichev et al., Science 260: 778 [1993], Johnson et al., Science 269 : 238 [1995]). In FIG. 20B, the cutting rates of Taq DN RX HT G418K / E507Q, Taq DN RX HT and Tth DN RX HT are plotted as a function of temperature. Black circles represent Taq DN RX HT enzyme. White circles represent Tth DN RX HT enzyme and Xs represents Taq DN RX HT G418K / E507Q enzyme. The difference in activity of Tth and Taq DN RX HT enzymes against IrT substrate is even greater than the difference in IL-6 RNA substrate when tested with the cleavage assay described in Example 1. The G418K / E507Q mutation increases the activity of the Taq enzyme more than 10 times, increasing by 25% compared to the Tth enzyme. All three enzymes show a typical temperature profile for the cleavage signal amplification reaction and the same optimum temperature. Under the same conditions, the G418K / E507Q mutation had no significant effect on the DNA-dependent 5 'nuclease activity of Taq DN RX HT in any of the DNA substrate analogues to IrT (SEQ ID NO: 168). .

The effects of KCl and MgSO ₄ concentrations on the 5 ′ nuclease activity of Taq DN RX HT G418K / E507Q, Taq DN RX HT and Tth DN RX HT on IrT substrate are shown in FIGS. 20C and D. The activity of both enzymes showed similar salt dependence, and the optimum KCl concentration was 100 mM for Taq DN RX HT G418K / E507Q and Tth DN RX HT and 50 mM for Taq DN RX HT. The optimum MgSO ₄ concentration for both enzymes was about 8 mM. According to the analysis of the data in Fig. 20, the properties of Taq DN RX HT G418K / E507Q are closer to those of Tth DN RX HT than Taq DN RX HT, and G418K / E507Q mutation is important in substrate recognition for RNA targets To occupy an important role.

Use an excess of IrT substrate (SEQ ID NO: 166) and downstream probe (SEQ ID NO: 167) to determine the mechanism by which 5 'nuclease activity is reduced in the presence of RNA versus DNA target, and a constant enzyme concentration The Michaelis constant (K _m ) and maximum catalyst rate (k _cat ) of these three enzymes were determined. For these measurements, the composition of 10 μl of the reaction solution was 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% nonidet P-40, 10 μg / ml tRNA, 4 mM MgCl ₂ , 1 nM enzyme (Taq DN RX HT , Tth DN RX HT or Taq DN RX HT G418K / E507Q) and an equimolar mixture of IrT target and downstream probe at different concentrations (0.125 μM, 0.25 μM and 0.5 μM or 1 μM). The kinetics of cleavage at each enzyme and each substrate concentration was measured at 46 ° C. The reaction was stopped by adding 10 μl of 95% formamide containing 10 mM EDTA and 0.02% methyl violet (Sigma). 1 μl each of the stopped digestion reaction solution was fractionated using a 20% denaturing acrylamide gel (crosslinking 19: 1) containing 7 M urea in 45 mM Tris-borate, pH 8.3, 1.4 mM EDTA buffer. The gel was scanned on a FMBIO-100 fluorescent gel scanner (Hitachi) using a 585 nm filter. Fractions of the cleavage products (determined from the intensity of the band corresponding to the non-cleaved and cleaved substrate using FMBIO analysis software version 6.0 (Hitachi)) were plotted as a function of reaction time. Determined from the linear portion of the kinetics, the cleavage product concentration was defined as the enzyme concentration and reaction time (in minutes), the Michaelis constant K _m for each enzyme versus the maximum catalytic rate k _cat for the IrT substrate. It was determined from a plot of initial cutting speed as a function of concentration.

These three enzymes are similar K _m values have and k _cat values (range 200~300nM), Taq DN RX HT and Tth DN approximately 4 min ^-1 with respect RXHT, with respect to Taq DN RX HT G418K / E507Q Was found to be approximately 9 min- ¹ . The G418K / E507Q mutation increases Taq DN RX HT k _cat more than 2-fold, but has little effect on K _m , rather than placing the substrate in the proper position by cleavage by the mutation. It simply suggests increasing the binding constant.

Example 7
Using molecular modeling to further improve RNA-dependent 5 'nuclease activity
A. Point mutants To obtain enzymes with altered functions, mutations were introduced into sequences by site-directed mutagenesis or random mutagenesis at predetermined positions. As a result of the chimera test, the location of site-directed mutagenesis was selected based on the relevant disclosure literature and molecular modeling. From the two Tth DN RX HT enzymes and Taq DN RX HT enzymes described above, seven additional mutant enzymes and twenty additional mutant enzymes were prepared, respectively. Some mutant enzymes were obtained by multiple mutagenesis reactions. That is, two or more mutations were introduced to obtain the final product. Unless otherwise specified, mutation reactions were performed using the Tth DN RX HT construct described in Example 2C2 (SEQ ID NO: 273) or the Taq DN RX HT construct described in Example 2C1 (SEQ ID NO: 275). To obtain sufficient starting material for any mutagenesis reaction, plasmid DNA was purified from 200 ml JM109 cultured overnight using the QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.) According to the manufacturer's protocol. All site-specific mutations were introduced using the Transformer site-directed mutagenesis kit (Clontech) according to the manufacturer's protocol. For all mutagenesis reactions described, one of two different selection primers, trans-oligo AlwNI / SpeI or switch oligo SpeI / AlwNI (Clontech, Palo Alto CA catalog number 6488-1 or catalog number 6373-1) Either one was used. The selection oligo utilized for a given reaction depends on the choice of restriction enzyme sites in the vector. Both site-directed mutagenesis and random mutagenesis primers were synthesized using standard synthetic chemistry. Colonies obtained for each reaction were expressed in E. coli strain JM109. The random mutagenesis method is described below.

  Mutants were tested with the rapid screening protocol detailed in Example 1. The mutant protein was then expressed and purified as detailed in Example 3 when more detailed analysis was required or when larger scale protein preparation was required.

を用いて、pTrc99A Tth DN RX HT DNAの部位特異的変異誘発を実施し、H641A変異（DNA配列、配列番号：101；アミノ酸配列、配列番号：34）を導入した。あるいは、変異誘発用プライマー：

を用いて、H748A変異体（DNA配列、配列番号：102；アミノ酸配列、配列番号：35）を作製した。あるいは、変異誘発用プライマー：

を用いて、H786A変異体酵素（DNA配列、配列番号：103；アミノ酸配列、配列番号：36）を作製した。 1. Construction of Tth DN RX HT H641A, Tth DN RX HT H748A, Tth DN RX HT H786A Primers for mutagenesis:

Was used for site-directed mutagenesis of pTrc99A Tth DN RX HT DNA to introduce the H641A mutation (DNA sequence, SEQ ID NO: 101; amino acid sequence, SEQ ID NO: 34). Alternatively, mutagenesis primers:

Was used to prepare an H748A mutant (DNA sequence, SEQ ID NO: 102; amino acid sequence, SEQ ID NO: 35). Alternatively, mutagenesis primers:

Was used to prepare an H786A mutant enzyme (DNA sequence, SEQ ID NO: 103; amino acid sequence, SEQ ID NO: 36).

を用いて、部位特異的変異誘発を実施し、「TthAKK」（DNA配列、配列番号：104；アミノ酸配列、配列番号：37）と命名した変異体を作製した。 2. Construction of Tth DN RX HT (H786A / G506K / Q509K) Starting from the mutant Tth DN RX HT H786A prepared above, mutagenesis primers:

Was used to carry out site-directed mutagenesis to produce a variant named “TthAKK” (DNA sequence, SEQ ID NO: 104; amino acid sequence, SEQ ID NO: 37).

を用いて、部位特異的変異誘発反応を実施し、H784A変異を導入し、「Taq4M」（DNA配列、配列番号：105；アミノ酸配列、配列番号：38）と命名した変異体を作製した。 3. Construction of Taq DN RX HT (W417L / G418K / E507Q / H784A) Mutagenic oligonucleotide

Was used to perform a site-directed mutagenesis reaction, the H784A mutation was introduced, and a mutant named “Taq4M” (DNA sequence, SEQ ID NO: 105; amino acid sequence, SEQ ID NO: 38) was produced.

を用いて、Taq4M変異体に部位特異的変異誘発を実施し、Taq 4M H639A変異体（DNA配列、配列番号：106；アミノ酸配列、配列番号：39）を作製した。また、プライマー：

を用いて、Taq 4M R587A（DNA配列、配列番号：107；アミノ酸配列、配列番号：40）を作製した。また、プライマー：

を用いて、Taq 4M G504K（DNA配列、配列番号：108；アミノ酸配列、配列番号：41）を作製した。また、プライマー：

を用いて、Taq 4M G80E変異体（DNA配列、配列番号：109；アミノ酸配列、配列番号：42）を作製した。 4. Construction of Taq4M H639A, Taq4M R587A, Taq4M G504K and Taq4M G80E Primers:

Was used to perform site-directed mutagenesis of the Taq4M mutant to produce a Taq4M H639A mutant (DNA sequence, SEQ ID NO: 106; amino acid sequence, SEQ ID NO: 39). Also primers:

Was used to prepare Taq 4M R587A (DNA sequence, SEQ ID NO: 107; amino acid sequence, SEQ ID NO: 40). Also primers:

Was used to prepare Taq 4M G504K (DNA sequence, SEQ ID NO: 108; amino acid sequence, SEQ ID NO: 41). Also primers:

Was used to produce Taq 4M G80E mutant (DNA sequence, SEQ ID NO: 109; amino acid sequence, SEQ ID NO: 42).

を用いて、部位特異的変異誘発を実施し、P88E／P90E変異（DNA配列、配列番号：110；アミノ酸配列、配列番号：43）を作製した。また、プライマー：

を用いて、L109F／A110T変異（DNA配列、配列番号：111；アミノ酸配列、配列番号：44）を作製した。 5. Construction of Taq 4M P88E / P90E and Taq 4M L109F / A110T Starting from the above Taq 4M, primers for site-directed mutagenesis:

Was used to perform site-directed mutagenesis to generate a P88E / P90E mutation (DNA sequence, SEQ ID NO: 110; amino acid sequence, SEQ ID NO: 43). Also primers:

Was used to make the L109F / A110T mutation (DNA sequence, SEQ ID NO: 111; amino acid sequence, SEQ ID NO: 44).

を用いて、620塩基対のPCR断片が得られた。プライマー：

を用いて、別の510塩基対のPCR産物が得られた。この2種類のPCR産物は、外側のプライマー158-84-01および330-06-03を用いて最終的に組換えPCR増幅を実施すると1182塩基対産物が得られるような重複を有している。製造元の説明書に従い、組換えPCR産物を制限酵素 NotIおよびBamHIで消化して、793塩基対の断片を得た。また、元のプラスミド Taq4Mを同一の酵素で消化して、用いた連結用ベクターとして用いた。いずれのDNA断片も、連結前に、TAEアガロースゲルによって精製した。断片をベクターに連結し、JM109 細胞を形質転換した。このようにして、変異G499R、A502K、I503LおよびE507K、ならびに制限エンドヌクレアーゼ部位、AgeIを導入した。この構築物を「Taq 8M」（DNA配列、配列番号：112；アミノ酸配列、配列番号：45）と命名した。 6. Construction of Taq DN RX HT (W417L / G418K / G499R / A502K / I503L / G504K / E507K / H784A) First, two types of PCR using the construct Taq4M (Taq W417L / G418K / G504K / E507Q / H784A) as a template The reaction was carried out. Primer:

Was used to obtain a 620 base pair PCR fragment. Primer:

Was used to obtain another 510 base pair PCR product. The two PCR products overlap so that the final PCR amplification using the outer primers 158-84-01 and 330-06-03 yields a 1182 base pair product. . The recombinant PCR product was digested with restriction enzymes NotI and BamHI according to the manufacturer's instructions to obtain a 793 base pair fragment. The original plasmid Taq4M was digested with the same enzyme and used as the ligation vector used. All DNA fragments were purified by TAE agarose gel before ligation. The fragment was ligated into the vector and transformed into JM109 cells. In this way, mutations G499R, A502K, I503L and E507K and a restriction endonuclease site, AgeI, were introduced. This construct was named “Taq 8M” (DNA sequence, SEQ ID NO: 112; amino acid sequence, SEQ ID NO: 45).

B. Random mutagenesis A number of enzymes with altered function were generated by random mutagenesis. Protein regions targeted for random mutagenesis were selected based on molecular modeling data and literature information. Mutations were introduced into different regions of the protein using different mutagenesis primers. Taq 4M G504K (Taq DN RX HT W417L / G418K / G504K / E507Q / H784A /) (SEQ ID NO: 108) was subjected to random mutagenesis, and Taq8M (sequence) unless otherwise specified. The homologous region of the mutant PCR fragment prepared by mutagenesis reaction was exchanged for No. 112).

  In addition, random mutagenesis was performed on the above Tth DN RX HT H786A (SEQ ID NO: 103). The mutant PCR fragment homology region prepared from the Tth DN RX HT H786A template was replaced with that of the original Tth DN RX HT H786A.

を、

と共に用いて、Taq ポリメラーゼ変異体Taq DN RX HT W417L／G418K／G504K／E507Q／H784A（配列番号：108）のアミノ酸の500位から507位にランダムな残基を導入した。括弧内の塩基の91％のみが不変であり、残りの9％の塩基が他の3種類の塩基の混合物となるようにプライマー 535-054-01を合成することによって、これを達成した。このオリゴの最初の不変な配列は、G499R、A502KおよびQ507K変異を含む。 1. Random mutants at amino acid residues 500-507 or 513-520 First mutant oligonucleotide:

The

In addition, random residues were introduced from position 500 to position 507 of the amino acid of the Taq polymerase mutant Taq DN RX HT W417L / G418K / G504K / E507Q / H784A (SEQ ID NO: 108). This was achieved by synthesizing primer 535-054-01 so that only 91% of the bases in parenthesis were unchanged and the remaining 9% bases were a mixture of the other three bases. The first invariant sequence of this oligo contains the G499R, A502K and Q507K mutations.

  In order to introduce mutations in the region from 500 to 507, primers 535-054-01 and primers 158-84-01, Advantage cDNA PCR kit (Clonetech), and the above Taq variants were used for PCR reactions. . The PCR fragment was then run on a 1% TAE agarose gel, excised and purified with the QIAquick Gel Extraction kit (Qiagen, Valencia CA, catalog number 28706). The purified fragment was cut with NotI and AgeI and ligated into pTaq8M linearized with NotI and AgeI. JM109 E. coli cells (Promega) were transformed with the resulting product. Clones were tested as described below.

を、

と共に用いて、アミノ酸513-520のランダムな残基に導入した。また、プライマー535-054-02の括弧内の塩基は、91％のみが野性型で、おのおの3％が残りの3種類のヌクレオチドである。 Second mutant oligonucleotide (used in different reactions):

The

Used in conjunction with random residues at amino acids 513-520. Furthermore, only 91% of the bases in parentheses of primer 535-054-02 are wild type, and 3% of each is the remaining three nucleotides.

  For the purpose of introducing mutation into the region of 513-520, primer 535-054-02 and primer 535-054-02, and the above-mentioned Taq DN RX HT W417L / G418K / G504K / E507Q / H784A (SEQ ID NO: 108) as a template ) Was used in the PCR reaction. As described above, the obtained PCR fragment was purified and digested with restriction enzymes AgeI and BamHI. The cleaved fragment was then ligated to the Taq8M construct linearized with AgeI and BamHI. JM109 E. coli cells were transformed with the ligation product. Clones were tested as described in Example 1. The mutants produced from these are as follows. Taq DN RX HT W417L / G418K / G499R / A502K / K504N / E507K / H784A (Ml-13) (DNA sequence, SEQ ID NO: 113; amino acid sequence, SEQ ID NO: 46). Taq DN RX HT W417L / G418K / G499R / L500I / A502K / G504K / Q507H / H784A (Ml-36) (DNA sequence, SEQ ID NO: 114; amino acid sequence, SEQ ID NO: 47). Taq DN RX HT W417L / G418K / G499R / A502K / I503L / G504K / E507K / T514S / H784A. (DNA sequence, SEQ ID NO: 115; amino acid sequence, SEQ ID NO: 48). Taq DN RX HT W417L / G418K / G499R / A502K / I503L / G504K / E507K / V518L / H784A (M2-06) (DNA sequence, SEQ ID NO: 116; amino acid sequence, SEQ ID NO: 49).

2. TthDN RX HT H786A random mutagenesis method
TthDN RX HT H786A (SEQ ID NO: 36) In order to create a mutant of the helix-hairpin-helix region of the enzyme, two different PCR above reactions were performed using the H786A (SEQ ID NO: 103) mutant as a template. did. Recombinant PCR Two PCR products are overlapped so that the above reaction can be carried out (Higuchi, “PCR Technology”, edited by HA Eriich, Stockton Press, New York. Pp6l-70 [1989]). This final PCR product was then exchanged with the homologous region of the TthDN H786A mutant using the fragment ends and restriction enzyme sites located within the TthDN H786A sequence.

を用いて、620塩基対のPCR断片が得られた。Advantage cDNA PCRキット（Clontech）を用いて製造元の説明書に従い、PCR反応を実施した。このPCR産物は、アミノ酸1〜194位を含む。この反応によっては、変異は何も導入されないが、EcoRI制限酵素部位が5'末端に存在する。 Starting from TthDN H786A above, primers:

Was used to obtain a 620 base pair PCR fragment. PCR reaction was performed using Advantage cDNA PCR kit (Clontech) according to the manufacturer's instructions. This PCR product contains amino acids 1-194. In this reaction, no mutation is introduced, but an EcoRI restriction enzyme site is present at the 5 'end.

を用いて、787塩基対のPCR断片を作製した。PCR反応は上記のように実施した。この断片は、変異誘発用プライマー604-08-05によりランダム変異を含む。配列の91％が野性型で、残りの9％が残りの3種類の塩基を均等に有するように、このプライマーの括弧内の塩基を合成した。 Starting from the above TthDN RX HT H786A, mutagenesis primers:

Was used to prepare a 787 base pair PCR fragment. PCR reactions were performed as described above. This fragment contains random mutations due to mutagenesis primers 604-08-05. The bases in the brackets of this primer were synthesized so that 91% of the sequence was wild type and the remaining 9% had the remaining 3 bases equally.

  These two types of fragments have an overlap and were joined by a recombinant PCR reaction. Primers 390-76-08 and 209-74-02 were added and an Advantage cDNA PCR kit (Clontech) was used according to the manufacturer's instructions. A 1380 base pair product was generated in this reaction.

  According to the manufacturer's instructions, the recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI to obtain a 986 base pair fragment. TthDN RX HT H786A was prepared by cutting with the same enzyme. Next, the fragment was ligated into a vector, and JM109 cells were transformed. New variants generated from this reaction set include: TthDN RX HT H786A / P197R / K200R (DNA sequence, SEQ ID NO: 117; amino acid sequence, SEQ ID NO: 50). TthDN RX HT H786A / K205Y (DNA sequence, SEQ ID NO: 118; amino acid sequence, SEQ ID NO: 51). TthDN RX HT H786A / G203R (DNA sequence, SEQ ID NO: 119; amino acid sequence, SEQ ID NO: 52).

を適当な選択プライマーと共に用い、部位特異的変異誘発反応を実施して、L109FおよびA110T変異を導入し、この「TaqSS」（DNA配列、配列番号：120；アミノ酸配列、配列番号：53）と命名した酵素を作製した。 3. Construction of Taq DN RX HT W417L / G418K / H784A L109F / A110T / G499R / A502K / I503L / G504K / E507K / T514S (Taq SS) Taq DN RX HT W417L / G418K / G499R / A502K / I503L / G504K / Starting from E507K / T514S / H784A (SEQ ID NO: 115) mutant, primer:

Is used with an appropriate selection primer to perform a site-directed mutagenesis reaction to introduce the L109F and A110T mutations, termed this “TaqSS” (DNA sequence, SEQ ID NO: 120; amino acid sequence, SEQ ID NO: 53) The enzyme was made.

を適当な選択プライマーと共に用い、部位特異的変異誘発反応を実施し、P88EおよびP90E変異を導入して、この酵素（DNA配列、配列番号：121；アミノ酸配列、配列番号：54）を作製した。 4. Construction of Taq DN RX HT W417L / G418L / H784A P88E / P90E / G499R / A502K / I503L / G504K / E507K / T514S Taq DN RX HT W417L / G418K / G499R / A502K / I503L / G504K / E507K / T514S / Starting from the H784A (SEQ ID NO: 115) mutant, the primer:

Was used in conjunction with appropriate selection primers to perform site-directed mutagenesis reactions to introduce P88E and P90E mutations to make this enzyme (DNA sequence, SEQ ID NO: 121; amino acid sequence, SEQ ID NO: 54).

を利用して、PCR産物の一方は、断片にEcoRI部位を導入したが変異は導入しなかった。第二のPCR産物では、変異誘発用プライマー：

およびプライマー209-74-02（配列番号：316）を利用した。この断片は、ランダム点突然変異を含み、組換えPCRを第一の断片と組み合わせた場合には、制限酵素EcoRIおよびNotIで切断し、同様にEcoRIおよびNotIで切断したTaqSS構築物に連結した。次に、連結した構築物でJM109を形質転換した。下記のようにして、コロニーをスクリーニングした。この変異誘発で得た酵素は以下のものを含む。TaqSS K198N（DNA配列、配列番号：112；アミノ酸配列、配列番号：55）。TaqSS A205Q（DNA配列、配列番号：123；アミノ酸配列、配列番号：56）。TaqSS I200M／A205G（DNA配列、配列番号：124；アミノ酸配列、配列番号：57）。TaqSS K203N（DNA配列、配列番号：125；アミノ酸配列、配列番号：58）。TaqSS T204P（DNA配列、配列番号：126；アミノ酸配列、配列番号：59）。 5. TaqSS random mutagenesis Using random mutagenesis, additional changes are introduced into the helix-hairpin-helix domain of the TaqSS variant (SEQ ID NO: 120). Mutagenesis was performed by the method of Example 9 above. In the first step, two different PCR products with duplicates were generated. Oligo 390-76-08 (SEQ ID NO: 314) and

Using one, one of the PCR products introduced an EcoRI site into the fragment but not a mutation. In the second PCR product, mutagenesis primers:

And primer 209-74-02 (SEQ ID NO: 316) was used. This fragment contained a random point mutation and when recombinant PCR was combined with the first fragment, it was cut with the restriction enzymes EcoRI and NotI and ligated to the TaqSS construct that was also cut with EcoRI and NotI. Next, JM109 was transformed with the ligated construct. Colonies were screened as follows. Enzymes obtained by this mutagenesis include: TaqSS K198N (DNA sequence, SEQ ID NO: 112; amino acid sequence, SEQ ID NO: 55). TaqSS A205Q (DNA sequence, SEQ ID NO: 123; amino acid sequence, SEQ ID NO: 56). TaqSS I200M / A205G (DNA sequence, SEQ ID NO: 124; amino acid sequence, SEQ ID NO: 57). TaqSS K203N (DNA sequence, SEQ ID NO: 125; amino acid sequence, SEQ ID NO: 58). TaqSS T204P (DNA sequence, SEQ ID NO: 126; amino acid sequence, SEQ ID NO: 59).

を用いて、上記のように、部位特異的変異誘発を実施して、TaqSS R677A変異体（DNA配列、配列番号：127；アミノ酸配列、配列番号：60）を作製した。 6. Construction of TaqSS R677A Another specific point mutation was introduced into TaqSS in order to introduce sequence mutations in both the arch and polymerase regions of the enzyme. Oligo:

As described above, site-directed mutagenesis was performed to generate TaqSS R677A mutant (DNA sequence, SEQ ID NO: 127; amino acid sequence, SEQ ID NO: 60).

7. Construction of TaqTthAKK (DNA sequence, SEQ ID NO: 128; amino acid sequence, SEQ ID NO: 61) and TthTaq5M (DNA sequence, SEQ ID NO: 129; amino acid sequence, SEQ ID NO: 62)
Tth DN RX HT (H786A / G506K / Q509K) (SEQ ID NO: 104; abbreviated here as TthAKK) or Taq 4M G504 (SEQ ID NO: 108; abbreviated here as Taq 5M) is used as restriction endonuclease EcoRI and The chimeric mutants TaqTthAKK and TthTaq5M were generated by cutting with NotI. Smaller inserts and larger vector fragments were purified by gel as detailed in Example 3D. The insert was exchanged and ligated between the two mutants by the method described in Example 3D. Screening and confirmation of the construct sequence was performed in Example 3D.

Example 8
RNA-dependent 5 'nuclease activity of other polymerases
Using information obtained from TaqPoI / TthPol recombination, mutagenesis, and modeling, similar mutations were made for other DNA polymerases and their effects on the cleavage activity were examined. The thermus filiformis (TfiPol) and Thermus scottodactus (TscPol) DNA polymerases were cloned and purified by the method described in Example 2. The mutagenesis of these two proteins is described below.

以下の2種類のオリゴヌクレオチドを用いて、E507Q変異を導入した：

以下の2種類のオリゴヌクレオチドを用いて、D785N変異を導入した：

これら3種類の変異をすべて含むプラスミドを、pTrc99a-TfiPolDN2M（DNA配列、配列番号：130；アミノ酸配列、配列番号：63）と命名した。 A. Construction of TfiPolDN2M
Mutation of pTrc99a-TfiPol (SEQ ID NO: 249) was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The P420K mutation was introduced using two types of oligonucleotides:

The E507Q mutation was introduced using the following two oligonucleotides:

The following two oligonucleotides were used to introduce the D785N mutation:

A plasmid containing all these three types of mutations was named pTrc99a-TfiPolDN2M (DNA sequence, SEQ ID NO: 130; amino acid sequence, SEQ ID NO: 63).

以下の2種類のオリゴヌクレオチドを用いて、E505Q変異を導入した：

以下の2種類のオリゴヌクレオチドを用いて、D783N変異を導入した：

これら3種類の変異をすべて含むプラスミドを、pTrc99a-TscPolDN2M（DNA配列、配列番号：131；アミノ酸配列、配列番号：64）と命名した。 B. Construction of TscPolDN2M
Mutation of pTrc99a-TscPol (SEQ ID NO: 253) was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The E416K mutation was introduced using the following two oligonucleotides:

The E505Q mutation was introduced using the following two oligonucleotides:

The D783N mutation was introduced using the following two oligonucleotides:

A plasmid containing all these three types of mutations was named pTrc99a-TscPolDN2M (DNA sequence, SEQ ID NO: 131; amino acid sequence, SEQ ID NO: 64).

を用いて、Tfi DN 2Mのアミノ酸の1位にEcoRI部位およびアミノ酸の331位にNotI 部位を導入した。変異誘発用プライマー：

を用いて、Tsc DN 2Mのアミノ酸の1位にEcoRI部位およびアミノ酸の327位にNotI 部位を導入した。Advance cDNA PCRキット（Clonetech）を用いて製造元の説明書に従い、標的としてTfi DN 2Mまたは Tsc DN 2Mを用いて対応するプライマーでPCR反応を行った。1017塩基対のPCR産物をEcoRIおよびNotIの両方で切断し、実施例3Dの方法でゲルによって精製した993塩基対の挿入断片を得た。また、変異体 Taq4M G504K（配列番号：108）およびTm DN RX HT（H786A／G506K／Q509K）（配列番号：104）をEcoRIおよびNotIで切断し、より大きいベクター断片を上記のようにゲルで単離した。実施例3Dに詳述される方法で、ライゲーションを行い、新しい構築物のスクリーニングおよび確認を実施した。 C. Chimera of Tsc, Tfi, Tth and Taq variants
1. TfiTth AKK (DNA sequence, SEQ ID NO: 132; amino acid sequence, SEQ ID NO: 65), TscTthAKK (DNA sequence, SEQ ID NO: 133; amino acid sequence, SEQ ID NO: 66), TfiTaq5M (DNA sequence, SEQ ID NO: 134) Amino acid sequence, SEQ ID NO: 67) and TscTaq5M (DNA sequence, SEQ ID NO: 135; amino acid sequence, SEQ ID NO: 68)
Tth DN RX HT (H86A / G506K / Q509K) (abbreviated here as TthAKK, SEQ ID NO: 104) or Taq 4M G504 (abbreviated here as Taq 5M; SEQ ID NO: 108) and Tfi DN 2M (sequence No. 130) or Tsc DN 2M (SEQ ID NO: 131) for the purpose of creating a chimeric enzyme, another restriction endonuclease site was introduced into the variants named Tfi and Tsc by site-directed mutagenesis. . Mutagenesis primer:

Was used to introduce an EcoRI site at position 1 of the amino acid of Tfi DN 2M and a NotI site at position 331 of the amino acid. Mutagenesis primers:

Was used to introduce an EcoRI site at position 1 of the amino acid of Tsc DN 2M and a NotI site at position 327 of the amino acid. A PCR reaction was performed with corresponding primers using Tfi DN 2M or Tsc DN 2M as a target using Advance cDNA PCR kit (Clonetech) according to the manufacturer's instructions. The 1017 base pair PCR product was cut with both EcoRI and NotI to obtain a 993 base pair insert purified by gel with the method of Example 3D. Mutants Taq4M G504K (SEQ ID NO: 108) and Tm DN RX HT (H786A / G506K / Q509K) (SEQ ID NO: 104) were cut with EcoRI and NotI, and the larger vector fragment was isolated on a gel as described above. Released. Ligation was performed and new constructs were screened and confirmed as detailed in Example 3D.

Example 9
Another enzyme with improved RNA-dependent 5 'nuclease activity
To facilitate the subsequent cloning process of Tfi DN 2M (ΔN), a unique NotI site is removed from the polymerase region of the TfiPolDN2M gene (SEQ ID NO: 130 described in Example 8A) by removing the endogenous restriction enzyme site (NotI). Was inserted in a more advantageous position.

とQUIKCHANGE 部位特異的変異誘発キット（Stratagene）用い、製造元の説明書に従って実施した。新しい構築物を、Tfi DN 2M（ΔN）（DNA配列、配列番号：340；アミノ酸配列、配列番号：341）と命名した。 The endogenous NotI site was removed as follows. Mutagenesis primers:

And a QUIKCHANGE site-directed mutagenesis kit (Stratagene) and performed according to the manufacturer's instructions. The new construct was named Tfi DN 2M (ΔN) (DNA sequence, SEQ ID NO: 340; amino acid sequence, SEQ ID NO: 341).

と、鋳型としてTfi DN 2M（ΔN）（配列番号：340）をPCR上記反応で用いた。得られたPCR断片を精製し、制限酵素 NotIおよびSalIで切断した。次に、切断した断片を、NotIおよびSalIで消化して構築物 TfiTthAKK（実施例8Cに記載の配列番号：132）に連結した。新しい構築物は、Tfi DN 2M（N）（DNA配列、配列番号：345；アミノ酸配列、配列番号：346）と命名した。 Preparation of Tfi DN 2M (N) and Tsc DN 2M (N)
For the purpose of introducing a NotI site (position 328 of amino acid) unique to Tfi DN 2M (ΔN) (SEQ ID NO: 340):

Tfi DN 2M (ΔN) (SEQ ID NO: 340) was used in the above PCR reaction as a template. The obtained PCR fragment was purified and cleaved with restriction enzymes NotI and SalI. The cleaved fragment was then digested with NotI and SalI and ligated to the construct TfiTthAKK (SEQ ID NO: 132 as described in Example 8C). The new construct was named Tfi DN 2M (N) (DNA sequence, SEQ ID NO: 345; amino acid sequence, SEQ ID NO: 346).

と、鋳型としてTscPolDN2M（配列番号：131）を用いてPCRを実施した。次に、PCR産物をNotI-SalIで消化した断片を、NotIおよびSalIで消化したTscTmAKK ベクター（配列番号：133）にサブクローニングした。得られた構築物を、Tsc DN 2M（N）（DNA配列、配列番号：347；アミノ酸配列、配列番号：348）と命名した。 For the purpose of introducing a NotI restriction endonuclease site into the mutant TscPol DN 2M (described in Example 8B above), primers:

PCR was performed using TscPolDN2M (SEQ ID NO: 131) as a template. Next, a fragment obtained by digesting the PCR product with NotI-SalI was subcloned into a TscTmAKK vector (SEQ ID NO: 133) digested with NotI and SalI. The resulting construct was named Tsc DN 2M (N) (DNA sequence, SEQ ID NO: 347; amino acid sequence, SEQ ID NO: 348).

を用いて、Tfi 2M（N）AKK変異体（DNA配列、配列番号：358；アミノ酸配列、配列番号：359）を作製した。この構築物を「TfiAKK」と命名した。 Preparation of Tfi DN 2M (N) AKK and Tsc DN2M (N) AKK To introduce the “AKK” mutation set (G504K / E507K / H784A / D785N), the Tfi DN 2M (N) mutant (DNA sequence, SEQ ID NO: : 345) site-directed mutagenesis was performed. Primer:

Was used to prepare a Tfi 2M (N) AKK variant (DNA sequence, SEQ ID NO: 358; amino acid sequence, SEQ ID NO: 359). This construct was named “TfiAKK”.

を用いて、Tsc2M（N）AKK（DNA配列、配列番号：346；アミノ酸配列、配列番号：365）を作製した。この構築物を、「Tsc AKK」と命名した。 In order to introduce the “AKK” mutation set (G502K / E505K / H782A / D783N) into the TscDN 2M (N) construct (DNA sequence, SEQ ID NO: 347), primers:

Was used to prepare Tsc2M (N) AKK (DNA sequence, SEQ ID NO: 346; amino acid sequence, SEQ ID NO: 365). This construct was named “Tsc AKK”.

およびプライマー：

をPCR反応に用いて、787塩基 対の断片を調製した。プライマー:

と、同一鋳型を反応に用いて別のPCR断片を得た。 Construction of point mutants by recombinant PCR
Construction of TthAKK (P195A) and TthAKK (P195K)
Mutagenesis primers for the purpose of introducing mutations at amino acid position 195 (P195A or P195K) in the nuclease domain of the TthAKK construct:

And primers:

Was used in a PCR reaction to prepare a 787 base pair fragment. Primer:

And another PCR fragment was obtained using the same template for the reaction.

  These two types of fragments have an overlap and were joined by a recombinant PCR reaction. Outer primers 390-076-08 and 209-074-02 were added and an Advantage cDNA PCR kit (Clontech) was used according to the manufacturer's instructions. This reaction produced a 1380 base pair product.

  The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI to obtain a 986 base pair fragment. A TthAKK construct was prepared by cutting with the same enzyme. Next, the fragment was vector ligated and JM109 cells were transformed. Novel mutants generated from this reaction set include TthAKK (P195A) (DNA sequence, SEQ ID NO: 373; amino acid sequence, SEQ ID NO: 374) and TthAKK (P195K) (DNA sequence, SEQ ID NO: 375; amino acid sequence, SEQ ID NO: 376).

2種類の重複PCR断片を生成させた。該2種類の産物を組み合わせ、外側のプライマー700-10-03および158-084-01で増幅した。組換えPCR産物を、制限酵素 NotIおよびBamHIで切断し、NotI／BamHIで予め切断した TthAKK構築物に連結した。この変異体を、TthAKK（N417K／L418K）（DNA配列、配列番号：379；アミノ酸配列、配列番号：380）と命名した。 Construction of TthAKK (N417K / L418K) TthAKK (N417K / L418K) was constructed using the same method. Mutagenesis primers:

Two overlapping PCR fragments were generated. The two products were combined and amplified with outer primers 700-10-03 and 158-084-01. The recombinant PCR product was cut with the restriction enzymes NotI and BamHI and ligated to the TthAKK construct previously cut with NotI / BamHI. This mutant was named TthAKK (N417K / L418K) (DNA sequence, SEQ ID NO: 379; amino acid sequence, SEQ ID NO: 380).

を用いて、TthAKK構築物に部位特異的変異誘発を実施し、TthAKK（P255L）（DNA配列、配列番号：383；アミノ酸配列、配列番号：384）を作製した。 Construction of another TthAKK point mutant by site-directed mutagenesis
Construction of TthAKK (P255L) Primer for mutagenesis:

Was used to perform site-directed mutagenesis on the TthAKK construct to generate TthAKK (P255L) (DNA sequence, SEQ ID NO: 383; amino acid sequence, SEQ ID NO: 384).

を用いて、TthAKK構築物に部位特異的変異誘発を実施し、TthAKK（F311Y）（DNA配列、配列番号： 387；アミノ酸配列、配列番号： 388）を作製した。 Construction of TthAKK (F311Y) Primer for mutagenesis:

Was used to perform site-directed mutagenesis on the TthAKK construct to generate TthAKK (F311Y) (DNA sequence, SEQ ID NO: 387; amino acid sequence, SEQ ID NO: 388).

を用いて、TthAKK構築物に部位特異的変異誘発を実施し、TthAKK（N221H／R224Q）（DNA配列、配列番号：391；アミノ酸配列、配列番号：392）を作製した。 Construction of TthAKK (N221H / R224Q) Primer for mutagenesis:

Was used to perform site-directed mutagenesis on the TthAKK construct to generate TthAKK (N221H / R224Q) (DNA sequence, SEQ ID NO: 391; amino acid sequence, SEQ ID NO: 392).

を用いて、TfhAKK構築物に部位特異的変異誘発を実施し、TthAKK（R251H）（DNA配列、配列番号：395；アミノ酸配列、配列番号：396）を作製した。 Construction of TthAKK (R251H) Mutagenesis primer:

Was used to perform site-directed mutagenesis on the TfhAKK construct to generate TthAKK (R251H) (DNA sequence, SEQ ID NO: 395; amino acid sequence, SEQ ID NO: 396).

を用いて、TthAKK（P255L）構築物に部位特異的変異誘発を実施し、TthAKK（P255L／R251H）（DNA配列、配列番号：399；アミノ酸配列、配列番号：400）を作製した。 Construction of TthAKK (P2S5L / R251H) Mutagenesis primer:

Was used to perform site-directed mutagenesis on the TthAKK (P255L) construct to generate TthAKK (P255L / R251H) (DNA sequence, SEQ ID NO: 399; amino acid sequence, SEQ ID NO: 400).

およびリバースプライマー：

を使用して、Tth AKK構築物上にパーム領域のランダム変異誘発を実施し、パーム領域にランダム変異を導入した。ブラケットは、表記の91％の塩基と3％のその他すべての塩基の合成を示す。 Construction of Tth AKK L429V (DNA sequence, SEQ ID NO: 401; amino acid sequence, SEQ ID NO: 402) Primer for mutagenesis:

And reverse primer:

Was used to perform random mutagenesis of the palm region on the Tth AKK construct, and random mutations were introduced into the palm region. Brackets indicate the synthesis of the indicated 91% base and 3% all other bases.

およびリバースプライマー：

を使用して、Tth AKK構築物上にパーム領域のランダム変異誘発を実施し、パーム領域にランダム変異を導入した。ブラケットは、表記の91％の塩基と3％のその他すべての塩基の合成を示す。 Construction of Tth AKK E425V (DNA sequence, SEQ ID NO: 405; amino acid sequence, SEQ ID NO: 406) Primer for mutagenesis:

And reverse primer:

Was used to perform random mutagenesis of the palm region on the Tth AKK construct, and random mutations were introduced into the palm region. Brackets indicate the synthesis of the indicated 91% base and 3% all other bases.

およびリバースプライマー：

を使用して、Tth AKK構築物上にパーム領域のランダム変異誘発を実施し、パーム領域にランダム変異を導入した。ブラケットは、表記の91％の塩基と3％のその他すべての塩基の合成を示す。 Construction of TthAKKL422N / E425K (DNA base sequence number: 407; amino acid sequence, sequence number: 408) Primer for mutagenesis:

And reverse primer:

Was used to perform random mutagenesis of the palm region on the Tth AKK construct, and random mutations were introduced into the palm region. Brackets indicate the synthesis of the indicated 91% base and 3% all other bases.

およびリバースプライマー：

を用いて、Tth AKK DNAパーム領域のランダム変異誘発を実施し、パーム領域にランダム変異を導入した。ブラケットは、表記の91％の塩基と3％のその他すべての塩基の合成を示す。 Construction of Tth AKK L422F / W430C (DNA sequence, SEQ ID NO: 409; amino acid sequence, SEQ ID NO: 410) Primer for mutagenesis:

And reverse primer:

Was used to perform random mutagenesis of the Tth AKK DNA palm region, and random mutations were introduced into the palm region. Brackets indicate the synthesis of the indicated 91% base and 3% all other bases.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、A504 アミノ酸にランダム変異を導入した。 Construction of Tth AKK A504F (DNA sequence, SEQ ID NO: 411; amino acid sequence, SEQ ID NO: 412) Mutagenesis primer:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations into the A504 amino acid.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、A504 アミノ酸にランダム変異を導入した。 Construction of Tth AKK A504V (DNA sequence, SEQ ID NO: 415; amino acid sequence, SEQ ID NO: 416) Mutagenesis primer:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations into the A504 amino acid.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、A504 アミノ酸にランダム変異を導入した。 Construction of Tth AKK A504S (DNA sequence, SEQ ID NO: 417; amino acid sequence, SEQ ID NO: 418) Mutagenesis primer:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations into the A504 amino acid.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、S517アミノ酸にランダム変異を導入した。 Construction of Tth AKK S517G (DNA sequence, SEQ ID NO: 419; amino acid sequence, SEQ ID NO: 420) Mutagenesis primer:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations at the S517 amino acid.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、A518アミノ酸にランダム変異を導入した。 Construction of Tth AKK A518L (DNA sequence, SEQ ID NO: 423; amino acid sequence, SEQ ID NO: 424) Primer for mutagenesis:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations at the A518 amino acid.

およびリバースプライマー：

を使用して、Tth AKK構築物上に部位特異的飽和変異誘発を実施し、A518アミノ酸にランダム変異を導入した。 Construction of Tth AKK A518R (DNA sequence, SEQ ID NO: 426; amino acid sequence, SEQ ID NO: 427) Primer for mutagenesis:

And reverse primer:

Was used to perform site-specific saturation mutagenesis on the Tth AKK construct to introduce random mutations at the A518 amino acid.

を使用して、Taq 5M構築物上に部位特異的変異誘発を実施し、Taq 5M 酵素にL451R変異を導入した。 Construction of Taq5M L451R (DNA sequence, SEQ ID NO: 428; amino acid sequence, SEQ ID NO: 429) Primer for mutagenesis:

Was used to perform site-directed mutagenesis on the Taq 5M construct to introduce the L451R mutation into the Taq 5M enzyme.

およびリバースプライマー：

を用いて、Tth AKK構築物上に部位特異的変異誘発を実施し、Tth AKK 酵素にA504K変異導入した。 Construction of Tth AKK A504K (DNA sequence, SEQ ID NO: 431; amino acid sequence, SEQ ID NO: 432) Primer for mutagenesis:

And reverse primer:

Was used to perform site-directed mutagenesis on the Tth AKK construct and introduce the A504K mutation into the Tth AKK enzyme.

およびリバースプライマー：

を用いて、Tth AKK構築物上に部位特異的変異誘発を実施し、Tth AKK 酵素にH641A変異導入した。 Construction of Tth AKK H641A (DNA sequence, SEQ ID NO: 435; amino acid sequence, SEQ ID NO: 436) Primer for mutagenesis:

And reverse primer:

Was used to perform site-directed mutagenesis on the Tth AKK construct and to introduce the H641A mutation into the Tth AKK enzyme.

およびリバースプライマー：

を用いて、Tth AKK構築物上に部位特異的変異誘発を実施し、Tth AKK 酵素にT508P変異導入した。 Construction of Tth AKK T508P (DNA sequence, SEQ ID NO: 439; amino acid sequence, SEQ ID NO: 440) Primer for mutagenesis:

And reverse primer:

Was used to perform site-directed mutagenesis on the Tth AKK construct and to introduce a T508P mutation into the Tth AKK enzyme.

Chimeras and mutations in chimeric fusions between the TthAKK enzyme and α-peptide Mutations that prevent expression of full-length fusion proteins based on colony blue-white screening (Wu et al., Nucleic Acids Research, 24: 1710 [1996]) A chimaeric fusion of TthAKK-lacZ-α-peptide was constructed for detection (including frameshift, deletion, insertion, etc.).

を用いて、TthAKK DNAに部位特異的変異誘発を実施し、TthAKKのC末端の6xHisタグの後のSalI部位にlacZ αペプチドが挿入されたpTthAKK-Lを調製した。プライマー：

を用いて、まず、pCRII-TOPO ベクター（Invitrogen）から、αペプチド（201アミノ酸を有する）をPCRで増幅した次に、PCR産物を、制限酵素SalIで消化し、同一の酵素で消化した pTthAKK-L ベクターに連結して、キメラ構築物 TthAKK-αペプチド（DNA配列、配列番号：481；アミノ酸配列、配列番号：482）を作製した。挿入物の方向をシーケンシングによって確認した。 Mutagenesis primers:

Was used to perform site-directed mutagenesis on TthAKK DNA to prepare pTthAKK-L in which the lacZ α peptide was inserted into the SalI site after the 6xHis tag at the C-terminal of TthAKK. Primer:

First, α peptide (having 201 amino acids) was amplified by PCR from pCRII-TOPO vector (Invitrogen), and then the PCR product was digested with restriction enzyme SalI and digested with the same enzyme pTthAKK- Ligated to the L vector, a chimeric construct TthAKK-α peptide (DNA sequence, SEQ ID NO: 481; amino acid sequence, SEQ ID NO: 482) was produced. The orientation of the insert was confirmed by sequencing.

Construction of TthTscAKK and TthTfiAKK enzymes
The TthAKK construct was cut with the enzymes EcoRI and NotI and the smaller insert was isolated on a gel. In addition, TscAKK or TfiAKK constructs were cut with EcoRI and NotI, and larger fragments were isolated on gels and purified. Tth insert (nuclease domain) was ligated to TscAKK and TfiAKK vectors (polymerase domain) and TthTscAKK (DNA sequence, SEQ ID NO: 447; amino acid sequence, SEQ ID NO: 448) and TthTfiAKK (DNA sequence, SEQ ID NO: 449; Amino acid sequence, SEQ ID NO: 450) A chimeric construct was made.

を用いて TaqTthAKK構築物に部位特異的変異誘発を実施して、L107F／A108T変異を導入し、Taq（FT）TthAKK（DNA配列、配列番号：484；アミノ酸配列、配列番号：485）を作製した。 Tth polymerase FT mutations to improve substrate specificity
Construction of Taq (FT) TthAKK Primer for mutagenesis:

Site-directed mutagenesis was carried out on the TaqTthAKK construct using A, and the L107F / A108T mutation was introduced to produce Taq (FT) TthAKK (DNA sequence, SEQ ID NO: 484; amino acid sequence, SEQ ID NO: 485).

を用いて、TfiTthAKK構築物に部位特異的変異誘発を実施して、L107F／V108T変異を導入し、Tfi（FT）TthAKK（DNA配列、配列番号：349；アミノ酸配列、配列番号：488）を作製した。 Construction of Tfi (FT) TthAKK Primer for mutagenesis:

Was used to introduce site-directed mutagenesis into the TfiTthAKK construct to introduce the L107F / V108T mutation to produce Tfi (FT) TthAKK (DNA sequence, SEQ ID NO: 349; amino acid sequence, SEQ ID NO: 488) .

Tfi (FT) DN 2M (N) and Tsc (FT) DN 2M (N) variants
Tfi (FT) TthAKK (DNA sequence, SEQ ID NO: 349; amino acid sequence, isolated from NotI and SalI fragments of Tfi DN 2M (N) variant (DNA sequence, SEQ ID NO: 345) and previously digested with NotI-SalI L107F / V108T mutation is introduced by inserting into SEQ ID NO: 488) to obtain a Tfi (FT) DN 2M (N) mutant (DNA sequence, SEQ ID NO: 350; amino acid sequence, SEQ ID NO: 351) It was. The same method was performed for the purpose of adding the L107F or E108T mutation to this Tsc-based construct. A fragment obtained by cleaving a Tsc DN 2M (N) mutant (DNA sequence, SEQ ID NO: 348) with NotI and SalI was converted into a Tsc (FT) TthAKK (DNA sequence, SEQ ID NO: 491) vector previously digested with NotI-SalI. Insertion was made to produce a Tsc (FT) DN 2M (N) mutant (DNA sequence, SEQ ID NO: 352; amino acid sequence, SEQ ID NO: 353).

を用いて、部位特異的変異誘発法により変異（H784A、G504KおよびE507K）の「AKK」セットを導入した。得られた変異体構築物は、Tfi（FT）AKK（DNA配列、配列番号：366；アミノ酸配列、配列番号：367）と命名した。 Construction of Tfi (FT) AKK and Tsc (FT) AKK Starting from the above Tfi (FT) DN2M (N) construct (DNA sequence, SEQ ID NO: 350), primers:

Was used to introduce the “AKK” set of mutations (H784A, G504K and E507K) by site-directed mutagenesis. The resulting mutant construct was named Tfi (FT) AKK (DNA sequence, SEQ ID NO: 366; amino acid sequence, SEQ ID NO: 367).

を用い、鋳型としてTsc（FT）DN2M（N）変異体（DNA配列、配列番号：352）を用いて、部位特異的変異誘発反応を実施し、Tsc（FT）AKK変異体（DNA配列、配列番号：368；アミノ酸配列、配列番号：369）を作製した。 Similarly, primers:

Using Tsc (FT) DN2M (N) mutant (DNA sequence, SEQ ID NO: 352) as a template, a site-directed mutagenesis reaction was performed, and Tsc (FT) AKK mutant (DNA sequence, sequence) was used. No: 368; amino acid sequence, SEQ ID NO: 369).

およびリバースプライマー：

を用いて、TscTthAKK構築物に部位特異的変異誘発を実施し、L107F／E108T変異を導入して、Tsc（FT）TthAKK（DNA配列、配列番号：454；アミノ酸配列、配列番号：491）を作製した。 Construction of Tsc (FT) TthAKK Primer for mutagenesis:

And reverse primer:

Was used to perform site-directed mutagenesis on the TscTthAKK construct and introduced the L107F / E108T mutation to produce Tsc (FT) TthAKK (DNA sequence, SEQ ID NO: 454; amino acid sequence, SEQ ID NO: 491) .

Construction of Taq (FT) TscAKK and Taq (FT) TfiAKK enzymes
The Taq (FT) TthAKK construct was cut with the enzymes EcoRI and NotI and the smaller insert was isolated on a gel. In addition, TscAKK or TfiAKK constructs were cut with EcoRI and NotI, and larger fragments were isolated on gels and purified. Taq (FT) insert (nuclease domain) was ligated to TscAKK and TfiAKK vectors (polymerase domain) and Taq (FT) TscAKK (DNA sequence, SEQ ID NO: 443; amino acid sequence, SEQ ID NO: 444) and Taq ( FT) TfiAKK (DNA sequence, SEQ ID NO: 445; amino acid sequence, SEQ ID NO: 446) chimeric construct was made.

を用いて、Taq（FT）-Tth（AKK） DNA（配列番号：484）に部位特異的変異誘発を実施し、K70E変異を導入した。 Construction of TaqEFT-Tth (AKK) (DNA sequence, SEQ ID NO: 501; amino acid sequence, SEQ ID NO: 502) Primer for mutagenesis:

Was used to perform site-directed mutagenesis on Taq (FT) -Tth (AKK) DNA (SEQ ID NO: 484) to introduce the K70E mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、G4EA変異を導入した。 Additional mutations to improve the enzymatic activity of Taq (EFT) TthAKK
Construction of TaqEFT-Tth (AKK) -A / Ml (DNA sequence, SEQ ID NO: 505; amino acid sequence, SEQ ID NO: 506) Mutagenesis primer:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce a G4EA mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、H29F変異を導入した。 Construction of TaqEFT-Tth (AKK) -B / M2 (DNA sequence, SEQ ID NO: 509; amino acid sequence, SEQ ID NO: 510) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce the H29F mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、K57R変異を導入した。 Construction of TaqEFT-Tth (AKK) -C / M3 (DNA sequence, SEQ ID NO: 513; amino acid sequence, SEQ ID NO: 514) Mutagenesis primer:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce the K57R mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、S125T変異を導入した。 Construction of TaqEFT-Tth (AKK) -D / M5 (DNA sequence, SEQ ID NO: 517; amino acid sequence, SEQ ID NO: 518) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce the S125T mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、R206L変異を導入した。 Construction of TaqEFT-Tth (AKK) -E / M6 (DNA sequence, SEQ ID NO: 521; amino acid sequence, SEQ ID NO: 522) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce the R206L mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、R269GおよびR271K変異を導入した。 Construction of TaqEFT-Tth (AKK) -F / M7 (DNA sequence, SEQ ID NO: 525; amino acid sequence, SEQ ID NO: 526) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce R269G and R271K mutations.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、E290G、S291G、P292E、A294PおよびL295R変異を導入した。 Construction of TaqEFT-Tth (AKK) -G / M8 (DNA sequence, SEQ ID NO: 529; amino acid sequence, SEQ ID NO: 530) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce E290G, S291G, P292E, A294P and L295R mutations.

を用いて、Taq（EFT）-Tm（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、A328C変異を導入した。 Construction of TaqEFT-Tth (AKK) -H / M9 (DNA sequence, SEQ ID NO: 533; amino acid sequence, SEQ ID NO: 534) Mutagenesis primer:

Was used for site-directed mutagenesis of Taq (EFT) -Tm (AKK) DNA (SEQ ID NO: 501) to introduce the A328C mutation.

を用いて、Taq（EFT）-Tth（AKK） DNA（配列番号：501）に部位特異的変異誘発を実施し、E210K変異を導入した。 Construction of TaqEFT-Tth (AKK) -I / M10 (DNA sequence, SEQ ID NO: 537; amino acid sequence, SEQ ID NO: 538) Primer for mutagenesis:

Was used for site-directed mutagenesis of Taq (EFT) -Tth (AKK) DNA (SEQ ID NO: 501) to introduce the E210K mutation.

を用いて、108塩基対の断片を得た。PCR反応2では：

を用いて、117塩基対の断片を得た。PCR反応3では：

を用いて、237塩基対の断片を得た。PCR反応4では：

を用いて、276塩基対の断片を得た。PCR反応5では：

を用いて、288塩基対の断片を得た。PCR反応6では：

を用いて、113塩基対の断片を得た。PCR反応7では：

を用いて、157塩基対の断片を得た。これら7種のPCR産物は、重複を有しており、プライマー非存在下でのPCR増幅により適当な1005塩基対の産物を生じ、K70E、G4EA、H29F、K57R、S125T、R206L、R269G、R271K、E290G、S291G、P292E、A294P、L295RおよびA328C変異が導入される。外側のプライマー: 1044-038-1（配列番号：507）および1044-038-18（配列番号：536）を用いて産物をさらに増幅し、pPCR-Script-Ampにクローニングした。プライマー: 1044-038-1（配列番号：507）および1044-038-18（配列番号：536）を用いて、この構築物から、再び、ヌクレアーゼ・ドメインを増幅し、EcoRIおよびNotIで消化した。消化したPCR産物を、EcoRIおよびNotIで消化したTaqEFT-Tth（AKK）構築物に連結し、タンパク質発現およびスクリーニングを目的として、JM109を形質転換した。 Using the construct TaqEFT-Tth (AKK) (SEQ ID NO: 501) as a construction template for TaqEFT-Tth (AKK) -Ml-9 (DNA sequence, SEQ ID NO: 541; amino acid sequence, SEQ ID NO: 542), and the following Seven different PCR reactions were performed using the mutagenesis primer pairs.
In PCR reaction 1:

Was used to obtain a 108 base pair fragment. For PCR reaction 2:

Was used to obtain a 117 base pair fragment. For PCR reaction 3:

Was used to obtain a 237 base pair fragment. For PCR reaction 4:

Was used to obtain a 276 base pair fragment. In PCR reaction 5:

Was used to obtain a 288 base pair fragment. For PCR reaction 6:

Was used to obtain a 113 base pair fragment. For PCR reaction 7:

Was used to obtain a 157 base pair fragment. These seven PCR products have duplication, and PCR amplification in the absence of primers yields an appropriate 1005 base pair product, K70E, G4EA, H29F, K57R, S125T, R206L, R269G, R271K, E290G, S291G, P292E, A294P, L295R and A328C mutations are introduced. The product was further amplified using outer primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and cloned into pPCR-Script-Amp. From this construct, the nuclease domain was again amplified and digested with EcoRI and NotI using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536). The digested PCR product was ligated to a TaqEFT-Tth (AKK) construct digested with EcoRI and NotI, and JM109 was transformed for protein expression and screening purposes.

を用いて、108塩基対の断片を得た。PCR反応2では：

を用いて、117塩基対の断片を得た。PCR反応3では：

を用いて、237塩基対の断片を得た。PCR反応4では：

を用いて、299塩基対の断片を得た。PCR反応5では：

を用いて、228塩基対の断片を得た。PCR反応6では：

を用いて、113塩基対の断片を得た。PCR反応7では：

を用いて、157塩基対の断片を得た。これら7種のPCR産物は、重複を有しており、プライマー非存在下でのPCR増幅により適当な1005塩基対の産物を生じ、K70E、G4EA、H29P、K57R、S125T、R206L、E210K、R269G、R271K、E290G、S291G、P292E、A294P、L295RおよびA328C変異が導入される。外側のプライマー: 1044-038-1（配列番号：507）および1044-038-18（配列番号：536）を用いて産物をさらに増幅し、pPCR-Script-Ampにクローニングした。プライマー: 1044-038-1（配列番号：507）および1044-038-18（配列番号：536）を用いて、この構築物から、再び、ヌクレアーゼ・ドメインを増幅し、EcoRIおよびNotIで消化した。消化したPCR産物を、EcoRIおよびNotIで消化したTaqEFT-Tth（AKK）構築物に連結し、タンパク質発現およびスクリーニングを目的として、JM109を形質転換した。 Construction of TaqEFT-Tth (AKK) -Ml-10 (DNA sequence, SEQ ID NO: 543; amino acid sequence, SEQ ID NO: 544) Using the construct TaqEFT-Tth (AKK) (SEQ ID NO: 501) as a template, the following Seven different PCR reactions were performed using the mutagenesis primer pairs. In PCR reaction 1:

Was used to obtain a 108 base pair fragment. For PCR reaction 2:

Was used to obtain a 117 base pair fragment. For PCR reaction 3:

Was used to obtain a 237 base pair fragment. For PCR reaction 4:

Was used to obtain a 299 base pair fragment. In PCR reaction 5:

Was used to obtain a 228 base pair fragment. For PCR reaction 6:

Was used to obtain a 113 base pair fragment. For PCR reaction 7:

Was used to obtain a 157 base pair fragment. These seven PCR products have overlapping, and PCR amplification in the absence of primers yields a suitable 1005 base pair product, K70E, G4EA, H29P, K57R, S125T, R206L, E210K, R269G, R271K, E290G, S291G, P292E, A294P, L295R and A328C mutations are introduced. The product was further amplified using outer primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536) and cloned into pPCR-Script-Amp. From this construct, the nuclease domain was again amplified and digested with EcoRI and NotI using primers: 1044-038-1 (SEQ ID NO: 507) and 1044-038-18 (SEQ ID NO: 536). The digested PCR product was ligated to a TaqEFT-Tth (AKK) construct digested with EcoRI and NotI, and JM109 was transformed for protein expression and screening purposes.

を用いて、TthAKK、TaqTthAKK、TfiTthAKKおよびTscTthAKK DNAに部位特異的変異誘発を実施し、K69E変異を導入して、Tth（K69E）AKK（DNA配列、配列番号：452；アミノ酸配列、配列番号：494）、Taq（K69E）ThAKK、（DNA配列、配列番号：495；アミノ酸配列、配列番号：496）、Tfi（K69E）TthAKK（DNA配列、配列番号：497；アミノ酸配列、配列番号：498）およびTsc（K69E）TthAKK（DNA配列、配列番号：499；アミノ酸配列、配列番号：500）変異体酵素を作製した。 K69E mutation of the FEN enzyme to further improve substrate specificity
Construction of Tth (K69E) AKK, Taq (K69E) TthAKK, Tfi (K69E) TthAKK and Tsc (K69E) TthAKK and variants Primers for mutagenesis:

Was used to perform site-directed mutagenesis on TthAKK, TaqTthAKK, TfiTthAKK and TscTthAKK DNA, introducing the K69E mutation, and Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452; amino acid sequence, SEQ ID NO: 494). ), Taq (K69E) ThAKK, (DNA sequence, SEQ ID NO: 495; amino acid sequence, SEQ ID NO: 496), Tfi (K69E) TthAKK (DNA sequence, SEQ ID NO: 497; amino acid sequence, SEQ ID NO: 498) and Tsc (K69E) TthAKK (DNA sequence, SEQ ID NO: 499; amino acid sequence, SEQ ID NO: 500) mutant enzyme was prepared.

および鋳型として Tth（K69E）AKK（DNA配列、配列番号：452）を用いた。またプライマー：

および鋳型としてTsc（K69E）TthAKK（DNA配列、配列番号：454）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクター TthAKKに連結し、Tsc（167-333）TthAKK構築物（DNA配列、配列番号：455；アミノ酸配列、配列番号：456）を得た。 Construction of Tsc (167-334) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. Also primer:

As a template, Tsc (K69E) TthAKK (DNA sequence, SEQ ID NO: 454) was used. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI, and ligated to the vector TthAKK prepared by cleaving with the same enzymes, and the Tsc (167-333) TthAKK construct (DNA sequence, SEQ ID NO: 455; amino acid sequence, sequence) Number: 456).

および鋳型としてTth（K69E）AKK（DNA配列、配列番号：452）を用いた。またプライマー：

および鋳型としてTsc（K69E）TthAKK（DNA配列、配列番号：454）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクター TthAKK に連結し、Tsc（222-334）TfhAKK構築物（DNA配列、配列番号：459；アミノ酸配列、配列番号：460）を得た。 Construction of Tsc (222-334) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. Also primer:

As a template, Tsc (K69E) TthAKK (DNA sequence, SEQ ID NO: 454) was used. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI, and ligated to the vector TthAKK prepared by cleaving with the same enzymes, and the Tsc (222-334) TfhAKK construct (DNA sequence, SEQ ID NO: 459; amino acid sequence, sequence) Number: 460).

および鋳型として Tth（K69E）AKK（DNA配列、配列番号：452）を用いた。またプライマー：

および鋳型として Tfi（K69E）TthAKK（DNA配列、配列番号：497）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクターTthAKK に連結し、Tfi（222-334）TthAKK構築物（DNA配列、配列番号：463；アミノ酸配列、配列番号：464）を得た。 Construction of Tfi (222-334) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. Also primer:

And Tfi (K69E) TthAKK (DNA sequence, SEQ ID NO: 497) was used as a template. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI, ligated to the vector TthAKK prepared by cleaving with the same enzymes, and Tfi (222-334) TthAKK construct (DNA sequence, SEQ ID NO: 463; amino acid sequence, sequence) No. 464).

および鋳型として Tth（K69E）AKK（DNA配列、配列番号：452）を用いた。またプライマー：

および鋳型として Tfi（K69E）TthAKK（DNA配列、配列番号：498）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクター TthAKK に連結し、Tfi（167-333）TthAKK構築物（DNA配列、配列番号：467；アミノ酸配列、配列番号：468）を得た。 Construction of Tfi (167-334) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. Also primer:

And Tfi (K69E) TthAKK (DNA sequence, SEQ ID NO: 498) was used as a template. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI, ligated to the vector TthAKK prepared by cleaving with the same enzymes, and Tfi (167-333) TthAKK construct (DNA sequence, SEQ ID NO: 467; amino acid sequence, sequence) No. 468).

および鋳型としてTth（K69E）AKK（DNA配列、配列番号：452）を用いた。またプライマー：

および鋳型としてTsc（K69E）TthAKK（DNA配列、配列番号：499）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクター TthAKK に連結し、Tsc（167-333）TthAKK構築物（DNA配列、配列番号：471；アミノ酸配列、配列番号：472）を得た。 Construction of Tsc (lll-334) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. Also primer:

As a template, Tsc (K69E) TthAKK (DNA sequence, SEQ ID NO: 499) was used. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI and ligated to the vector TthAKK prepared by cleaving with the same enzymes, and the Tsc (167-333) TthAKK construct (DNA sequence, SEQ ID NO: 471; amino acid sequence, sequence) No. 472).

および鋳型としてTsc（K69E）TthAKK（DNA配列、配列番号：499）を用いた。またプライマー：

および鋳型としてTth（K69E）AKK（DNA配列、配列番号：452）を用いた。2種の産物を組み合わせて、外側のプライマー390-76-08および209-074-02で増幅した。組換えPCR産物を制限酵素EcoRIおよびNotIで切断し、同一の酵素で切断して調製したベクター TthAKKに連結し、Tsc（l-167）TthAKK構築物（DNA配列、配列番号：475；アミノ酸配列、配列番号：476）を得た。 Construction of Tsc (l-167) TthAKK
Two overlapping PCR fragments were generated. Primer:

As a template, Tsc (K69E) TthAKK (DNA sequence, SEQ ID NO: 499) was used. Also primer:

As a template, Tth (K69E) AKK (DNA sequence, SEQ ID NO: 452) was used. The two products were combined and amplified with outer primers 390-76-08 and 209-074-02. The recombinant PCR product was cleaved with restriction enzymes EcoRI and NotI, and ligated to the vector TthAKK prepared by cleaving with the same enzymes, and the Tsc (l-167) TthAKK construct (DNA sequence, SEQ ID NO: 475; amino acid sequence, sequence) No. 476).

Modification of AfuFEN enzyme
Construction of pAfuFEN Plasmid pAfuFEN1 was prepared as described in US patent application Ser. No. 09 / 684,938 and WO 98/23774, both of which are hereby incorporated by reference in their entirety. Briefly, using a DNA XTRAX kit (Gull Laboratories, Salt Lake City, UT) from 1 vial (approximately 5 ml culture) of A. fulgidus from DSMZ (DSMZ # 4304) Then, genomic DNA was prepared according to the manufacturer's protocol. The final DNA pellet was resuspended in 100 μl TE (10 mM Tris-HC1, pH 8.0, 1 mM EDTA). PCR was performed with 1 μl microliter of DNA solution using ADVANTAGE cDNA PCR kit (Clonetech). PCR was performed according to the manufacturer's recommendations.

である。得られた断片のクローニングは、PfuFEN1 遺伝子に関する米国特許出願第5,994,069号（様々な目的で本明細書にその全文が組み入れられる）に記載される方法で実施し、プラスミド pTrc99-AFFENlを作製した。発現を目的として、精製を簡便に進めるためにヒスチジン・テールを付加しpTrc99-AFFENlを改変して、pTrcAfuHisプラスミドを構築した。このヒスチジン・テールを付加する目的で、標準PCRプライマーによる部位特異的変異誘発法を用いて、pTrc99-AFFENlコード領域の最後のアミノ酸コドンと停止コドン間に6残基のヒスチジンをコードする配列を挿入した。得られたプラスミドを pTrcAfuHisと命名した。次に、上記のようにしてタンパク質を発現させ、Ni^＋＋ 親和性 カラムに結合して精製した。 The 5 'end primer is complementary to the 5' end of the Afu FEN-1 gene, except that it includes a 1 base pair substitution that creates an NcoI site. The 3 ′ end primer is complementary to the 3 ′ end of the Afu FEN-1 gene downstream of the FEN-1 ORF, except that it contains a two base substitution that generates a SalI site. The sequences of the 5 ′ and 3 ′ end primers are respectively

It is. Cloning of the resulting fragment was performed as described in US Patent Application No. 5,994,069 for the PfuFEN1 gene, which is incorporated herein in its entirety for various purposes, to produce plasmid pTrc99-AFFENl. For the purpose of expression, a pTrcAfuHis plasmid was constructed by adding a histidine tail and modifying pTrc99-AFFENl for easy purification. To add this histidine tail, insert a sequence encoding 6 histidines between the last amino acid codon and stop codon of the pTrc99-AFFENl coding region using site-directed mutagenesis with standard PCR primers. did. The resulting plasmid was named pTrcAfuHis. The protein was then expressed as described above and purified by binding to a Ni ⁺⁺ affinity column.

およびプライマー:

を用いた。2種の産物を組み合わせて、外側のプライマー700-10-03および390-076-08で増幅した。組換えPCR産物を制限酵素NcoIおよびSalIで切断し、同一の酵素で切断して調製したAfuFEN構築物にライゲートし、 Afu（Y236A）構築物（DNA配列、配列番号：549；アミノ酸配列、配列番号：550）を得た。 Construction of Afn (Y236A), A46-5 Two overlapping PCR fragments were prepared from the template AfuFEN (SEQ ID NO: 556). This includes primers:

And primer:

Was used. The two products were combined and amplified with outer primers 700-10-03 and 390-076-08. The recombinant PCR product was cleaved with the restriction enzymes NcoI and SalI, ligated to the AfuFEN construct prepared by cleaving with the same enzymes, and the Afu (Y236A) construct (DNA sequence, SEQ ID NO: 549; amino acid sequence, SEQ ID NO: 550). )

およびプライマー：

を用いた。2種の産物を組み合わせて、外側のプライマー700-10-03および390-076-08で増幅した。組換えPCR産物を制限酵素NcoIおよびSalIで切断し、同一の酵素で切断して調製したAfuFEN構築物にライゲートし、 Afu（Y236R）構築物（DNA配列、配列番号：552；アミノ酸配列、配列番号：553）を得た。 Construction of Afu (Y236R), A56-9 Two overlapping PCR fragments were prepared from the template AfuFEN. This includes primers:

And primers:

Was used. The two products were combined and amplified with outer primers 700-10-03 and 390-076-08. The recombinant PCR product was cleaved with restriction enzymes NcoI and SalI, ligated to the AfuFEN construct prepared by cleaving with the same enzymes, and Afu (Y236R) construct (DNA sequence, SEQ ID NO: 552; amino acid sequence, SEQ ID NO: 553). )

2. Chimera of FEN enzyme and thermus polymerase derivative The following enzyme constructs combined the polymerase domain of the AfuFEN enzyme and the polymerase domain part of the thermus polymerase. These combinations were designed based on information obtained from molecular modeling.

を用いて作製した。第二の断片は、鋳型としてTthAKK（配列番号：558）を用い、プライマーとして

を用いて作製した。二つの産物は配列重複領域を含み、組み合わせて、組換えPCR反応で外側のプライマー390-076-08および390-076-09で増幅した。組換えPCR産物を制限酵素BspEIおよびSalIで切断し、同一の酵素で切断して調製したAfuFEN構築物に連結し、Afu336-Tth296（AKK）構築物（DNA配列、配列番号：559；アミノ酸配列、配列番号：560）を得た。 Construction of Afu336-Tth296 (AKK), AT1-1
Two overlapping PCR fragments were generated. The first fragment uses the AfuFEN construct (SEQ ID NO: 556) as a template and as a primer

It was produced using. The second fragment uses TthAKK (SEQ ID NO: 558) as a template and as a primer.

It was produced using. The two products contained a sequence overlap region and were combined and amplified with the outer primers 390-076-08 and 390-076-09 in a recombinant PCR reaction. The recombinant PCR product was cleaved with restriction enzymes BspEI and SalI, ligated to the AfuFEN construct prepared by cleaving with the same enzymes, and the Afu336-Tth296 (AKK) construct (DNA sequence, SEQ ID NO: 559; amino acid sequence, SEQ ID NO: : 560).

および鋳型としてAfuFEN（DNA配列、配列番号：556）を用い、第二のものには、プライマー:

および鋳型としてTthAKK（DNA配列、配列番号：558）を用いた。2種の産物を組み合わせて、外側のプライマー390-076-08および390-076-09で増幅した。組換えPCR産物を制限酵素BspEIおよびSalIで切断し、同一の酵素で切断して調製したベクター pAfuFEN にライゲートし、 Afu336-Tth296（AKK）構築物（DNA配列、配列番号：563；アミノ酸配列、配列番号：564）を得た。 Construction of Afu328-Tth296 (AKK), AT2-3
Two overlapping PCR fragments were generated. The first includes primers:

And AfuFEN (DNA sequence, SEQ ID NO: 556) as a template, the second includes primers:

And TthAKK (DNA sequence, SEQ ID NO: 558) was used as a template. The two products were combined and amplified with outer primers 390-076-08 and 390-076-09. The recombinant PCR product was cleaved with restriction enzymes BspEI and SalI, ligated to the vector pAfuFEN prepared by cleaving with the same enzymes, and Afu336-Tth296 (AKK) construct (DNA sequence, SEQ ID NO: 563; amino acid sequence, SEQ ID NO: : 564).

Construction of Afu336-Taq5M
The Taq5M construct (SEQ ID NO: 41) was cut with the enzymes NotI and SalI and the smaller insert was isolated on a gel. In addition, the Afu336-Tth296 (AKK) construct (DNA sequence, SEQ ID NO: 559) was cleaved with the same restriction enzymes, and a larger vector fragment was purified. Next, it was ligated to an insert (Taq5M polymerase domain) vector to produce an Afu336-Taq5M construct (DNA sequence, SEQ ID NO: 565; amino acid sequence, SEQ ID NO: 566).

Construction of Afii336-TaqDN
The TaqDNHT construct was cut with the enzymes NotI and SalI and the smaller insert was isolated on a gel. In addition, the Afu336-Tth296 (AKK) construct (DNA sequence, SEQ ID NO: 559) was cleaved with the same restriction enzymes, and a larger vector fragment was purified. Next, the insert (TaqDN polymerase domain) was ligated into a vector to create an Afu336-Taq5M construct (DNA sequence, SEQ ID NO: 567; amino acid sequence, SEQ ID NO: 568).

Random chimerization of thermus polymerase
Based on the principle of DNA shuffling (Volkov and Arnold, Methods in Enzymology 328: 456 [2000]), a number of enzymes whose function was altered by random mutagenesis were generated by random chimerization of thermus polymerase. The following method was used for the following chimera production.

を用いて、ランダム・キメラ化を実施する目的の遺伝子をPCR増幅し、おおよそ1.4kbpの鋳型を作製した。約2μgのDNA鋳型を同一の比率で混合し、次に15℃で約1分間DNaseI（0.33U）により消化して、サイズが50〜200bpの断片を調製した。4％アガロースゲルでDNA断片を精製し、QIAEXII ゲル抽出キット（QIAGEN）を用いて製造元の説明書に従い抽出した。断片の再アセンブリー反応（PCRプログラム：94℃3分、94℃30秒-52℃1分-72℃2分＋5秒／サイクルで40サイクル、72℃4分）を実施するために、精製した断片（10μl）を10μlの2xPCRプレ混合物液（5倍希釈、クローン化Pfu緩衝液、各0.4mMのdNTP、0.06U／μlのクローン化Pfu DNAポリメラーゼ, STRATAGENEカタログ番号600153、添付の緩衝液）に添加した。次に、ネステッド・プライマー対：

を利用して、再アセンブル産物（10倍希釈を1μl）をPCRで増幅した。これは、CLONTECH GC melt cDNA PCRキット（カタログ番号K1907-Y）を用いて、製造元の説明書に従って実施した。精製したPCR産物を制限酵素EcoRIおよびNotIで消化し、次に同一の酵素で切断して調製した TthAKK構築物に連結し、連結混合物でJM109 コンピテント細胞を形質転換し、酵素活性に関してコロニーをスクリーニングした。 Primer:

Was used to PCR-amplify the target gene for random chimerization to produce a template of approximately 1.4 kbp. About 2 μg of DNA template was mixed in the same ratio and then digested with DNaseI (0.33 U) at 15 ° C. for about 1 minute to prepare a fragment of 50-200 bp in size. The DNA fragment was purified on a 4% agarose gel and extracted according to the manufacturer's instructions using the QIAEXII gel extraction kit (QIAGEN). Purified fragments to perform fragment reassembly reactions (PCR program: 94 ° C 3 minutes, 94 ° C 30 seconds-52 ° C 1 minute-72 ° C 2 minutes + 5 seconds / cycle 40 cycles, 72 ° C 4 minutes) (10 μl) is added to 10 μl of 2xPCR premix solution (5-fold dilution, cloned Pfu buffer, 0.4 mM each dNTP, 0.06 U / μl cloned Pfu DNA polymerase, STRATAGENE catalog number 600153, attached buffer) did. Next, nested primer pairs:

Was used to amplify the reassembled product (1 μl of 10-fold dilution) by PCR. This was performed using a CLONTECH GC melt cDNA PCR kit (Cat. No. K1907-Y) according to the manufacturer's instructions. The purified PCR product was digested with restriction enzymes EcoRI and NotI, then ligated to a TthAKK construct prepared by cutting with the same enzymes, JM109 competent cells were transformed with the ligation mixture, and colonies were screened for enzyme activity .

1. Generation of random chimeras S26 and S36 The templates used to generate these chimeric enzymes were the nuclease domains of the TthAKK, TaqTthAKK, TscTthAKK and TfiTthAKK constructs. Based on the first activity screen, two clones showed improved activity. Next, the sequences of random chimeras S26 (DNA sequence, SEQ ID NO: 571; amino acid sequence, SEQ ID NO: 572) and S36 (DNA sequence, SEQ ID NO: 573; amino acid sequence, SEQ ID NO: 574) are determined. Released.

を用いて、pS26 DNAに部位特異的変異誘発を実施し、L107F／E108T変異を導入し、S26（FT）（DNA配列、配列番号：575；アミノ酸配列、配列番号：576）を作製した。 2. Introduction of L107F / E108T, L109F / V110T and K69E mutations to improve the substrate specificity of S26 and S36
Construction of S26 (FT) Primer for mutagenesis:

Was used for site-directed mutagenesis of pS26 DNA to introduce L107F / E108T mutation to produce S26 (FT) (DNA sequence, SEQ ID NO: 575; amino acid sequence, SEQ ID NO: 576).

を用いて、pS26 DNAに部位特異的変異誘発を実施し、L109F／V110T変異を導入し、S36（FT）（DNA配列、配列番号：577；アミノ酸配列、配列番号：578）を作製した。 Construction of S36 (FT) Primer for mutagenesis:

Was used for site-directed mutagenesis of pS26 DNA to introduce L109F / V110T mutation to produce S36 (FT) (DNA sequence, SEQ ID NO: 577; amino acid sequence, SEQ ID NO: 578).

を用いて、pS26 DNAに部位特異的変異誘発を実施し、K69E変異を導入し、S26（K69E）（DNA配列、配列番号：579；アミノ酸配列、配列番号：580）を作製した。 Construction of S26 (K69E) Primer for mutagenesis:

Was used for site-directed mutagenesis of pS26 DNA to introduce the K69E mutation to produce S26 (K69E) (DNA sequence, SEQ ID NO: 579; amino acid sequence, SEQ ID NO: 580).

を用いて、S26（FT）DNAに部位特異的変異誘発を実施し、K69E変異を導入し、S26（FT／K69E）（DNA配列、配列番号：581；アミノ酸配列、配列番号：582）を作製した。 Construction of S26 (FT / K69E) Primer for mutagenesis:

Is used to perform site-directed mutagenesis on S26 (FT) DNA, introduce K69E mutation, and create S26 (FT / K69E) (DNA sequence, SEQ ID NO: 581; amino acid sequence, SEQ ID NO: 582) did.

3. Yet another random chimera, N3D7, N1A12 N1C4 and N2C3
The templates used to make these chimeric enzymes were the nuclease domains of Tth (K69E) AKK, Taq (K69E) TthAKK, Tsc (K69E) TthAKK, and Tfi (K69E) TthAKK constructs. Based on the first activity screen, four clones were shown to have improved activity. Next, random chimera N3D7 (DNA sequence, SEQ ID NO: 583; amino acid sequence, SEQ ID NO: 584), N1A12 (DNA sequence, SEQ ID NO: 585; amino acid sequence, SEQ ID NO: 586), N1C4 (DNA sequence, sequence SEQ ID NO: 587; amino acid sequence, SEQ ID NO: 588) and N2C3 (DNA sequence, SEQ ID NO: 589; amino acid sequence, SEQ ID NO: 590) were determined and isolated, respectively.

Example 10
Testing for the presence of upstream oligonucleotides of the enzyme When using structure-specific nucleases for sequential invasive cleavage reactions, (1) in the absence of upstream oligonucleotides, and (2) upstream oligonucleotides and downstream In the absence of overlap between probe and oligonucleotide, it is preferred that the enzyme has little probe cleavage ability. Figures 26a-e represent multiple structures that can be used. Enzyme activity is examined for each of these types of structures. Structure a (FIG. 26a) shows the probe oligonucleotide aligned in parallel with the target site on the bacteriophage M13 DNA (M13 sequence in FIG. 26 is shown in SEQ ID NO: 217) in the absence of the upstream oligonucleotide. . Structure b (FIG. 26b) contains an upstream oligonucleotide that does not contain a region that overlaps with a labeled probe (labeled with an asterisk). In structures c, d, and e (FIGS. 26c-e), the upstream oligonucleotide contains 1, 3, or 5 nucleotide overlaps, respectively, relative to the downstream probe oligonucleotide, each of these structures being a suitable invasive cleavage Represents the structure. For each of these structures, the activity of the enzyme Pfu FEN-1 was tested. Each reaction was performed twice.

Each reaction was performed using 10 μl of 10 mM MOPS (pH 7.5) and 7.5 mM MgCl ₂ containing 0.05% Tween20 and Nonidet P-40, respectively. 1 μM 5′TET-labeled probe oligonucleotide 89-15-1 (SEQ ID NO: : 212), 50 nM upstream oligonucleotide (oligo 81-69-2 [SEQ ID NO: 216], oligo 81-69-3 [SEQ ID NO: 215], oligo 81-69-4 [SEQ ID NO: 214], oligo 81 -69-5 [SEQ ID NO: 213] or any upstream oligonucleotide), 1 femtomole M13 target DNA, 10 mg / ml tRNA and 10 ng Pfu FEN-1.

All components except enzyme and MgCl ₂ were mixed in a final volume of 8 μl and overlaid with 10 μl of CHILLOUT liquid wax. The sample was heated to a reaction temperature of 69 ° C. By adding Pfu FEN-1 and MgCl ₂ in 2μl volumes to initiate the reaction. After incubation at 69 ° C. for 30 minutes, the reaction was stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02% methyl violet. Immediately before electrophoresis, the sample was heated at 90 ° C for 1 minute and was subjected to a 20% denaturing acrylamide gel (crosslinked 19: 1) containing 7M urea in 45mM Tris-Boric acid (pH8.3), 1.4mM EDTA buffer. Electrophoresed. Next, the gel was analyzed with FMBIO-100 Hitachi FMBIO fluorescence imaging apparatus. The obtained image is shown in FIG.

  In FIG. 27, lane “a” contains the product generated in the reaction performed in the absence of upstream oligonucleotide (structure a), and lane “b” cleaves the probe / target duplex (structure b). Do not include upstream oligonucleotides. Lanes “c”, “d”, and “e” contain the products generated in the reactions performed with the upstream oligonucleotides that cleave the probe / target duplex, 1, 3, and 5 bases, respectively. The size of uncleaved probe and cleavage product (in nucleotides) is shown to the left of the image in FIG. When structure a and structure b were used, probe cleavage was not detectable. In contrast, cleavage products were produced when invasive cleavage structures (structures c-e) were used. This data shows that the Pfu FEN-1 enzyme requires an upstream overlapping oligonucleotide for specific cleavage of the probe.

  By examining whether it is possible to test an enzyme that cleaves structures a to e, it is possible to examine whether the enzyme can be suitably used for a continuous invasive cleavage reaction (the specific oligonucleotide sequence shown in FIGS. Those skilled in the art will appreciate that these structures are merely exemplary of suitable test structures). A preferred enzyme will cleave structure a and structure b little or no. In addition, the structures c to e are specifically cleaved (that is, a cleaved product having a size expected from the degree of overlap between the oligonucleotides used to form the invasive cleavage structure is generated).

Example 11
Use of the product of the first invasive cleavage reaction that allows an overall increase in sensitivity in the second invasive cleavage reaction As described in the “Description of the Invention” above, the product of the first invasive cleavage reaction is used. By performing the second invasive cutting and completing the cutting structure in the second reaction, the detection sensitivity of the invasive cutting reaction can be increased. In this example, the probe cleaved in the first invasive cleavage reaction is used to form the embedded INVADER oligonucleotide and target molecule utilized in the second invasive cleavage reaction as indicated. .

  Designed to form a target strand that contains an INVADER oligonucleotide that incorporates the product ("cleavage probe 1") when the first probe contains internal complementarity and is cleaved in a first invasive cleavage reaction . Hybridization to the newly shaped target / INVADER molecule provides a second probe that is cleaved at the site of interest in the reaction. The standard invasive cleavage assay described above was performed simultaneously to demonstrate signal gain due to the performance of sequential invasive cleavage.

および
10nMの上流オリゴ：

ならびに10から100アトモルのM13標的DNA、10mg／ml tRNAおよび10ngのPfu FEN-1を含む。容積7μlに酵素およびMgCl₂以外の全成分を混合して、10μlのCHILLOUT液体ワックスを重層した。試料を反応温度62℃まで加熱した。2μlの容積でPfu FEN-1およびMgCl₂を添加することによって、反応を開始した。62℃で30分間保温後、10μlの95％ホルムアミド、10mM EDTA、0.02％メチルバイオレットで反応を停止した。 Both reactions were performed in duplicate. Each standard (ie, non-continuous) invasive cleavage reaction was performed in 10 μl containing 10 mM MOPS (pH 7.5), 8 mM MgCl ₂ , 0.05% Tween20 and Nonidet P-40, respectively, and 1 μM 5 ′ fluorescein labeled probe oligo:

and
10 nM upstream oligo:

As well as 10 to 100 attomoles of M13 target DNA, 10 mg / ml tRNA and 10 ng of Pfu FEN-1. All components except enzyme and MgCl ₂ were mixed in a volume of 7 μl and overlaid with 10 μl of CHILLOUT liquid wax. The sample was heated to a reaction temperature of 62 ° C. By adding Pfu FEN-1 and MgCl ₂ in 2μl volumes to initiate the reaction. After incubation at 62 ° C. for 30 minutes, the reaction was stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02% methyl violet.

10nMの上流オリゴヌクレオチド：

ならびに1μMの5'フルオレセイン標識オリゴヌクレオチド 106-32（第二のプローブもしくは「プローブ 2」、5'Fl-TGTTTTGACCT CCA-3'；配列番号：593）、1から100アトモルのM13標的DNA、10mg／ml tRNAおよび10ngのPfu FEN-1を含む。容積8μlに酵素およびMgCl₂以外の全成分を混合して、10μlのCHILLOUT液体ワックスを重層した。試料を反応温度62℃まで加熱した。（この温度は、プローブ1が第一の標的にアニーリングする最適温度である）。2μlの容積でPfu FEN-1およびMgCl₂を添加することによって、反応を開始した。62℃で15分間保温後、温度を58℃に下げ（この温度は、この温度は、プローブ2が第二の標的にアニーリングする最適温度である）、試料をさらに15分間保温した。10μlの95％ホルムアミド、20mM EDTA、0.02％メチルバイオレットで反応を停止した。 Each successive invasive cleavage reaction consists of 1 μM 5 ′ fluorescein labeled oligonucleotide in 10 μl containing 10 mM MOPS (pH 7.5), 8 mM MgCl ₂ , 0.05% Tween20 and Nonidet P-40, respectively:

10 nM upstream oligonucleotide:

And 1 μM 5 ′ fluorescein labeled oligonucleotide 106-32 (second probe or “probe 2”, 5′Fl-TGTTTTGACCT CCA-3 ′; SEQ ID NO: 593), 1 to 100 attomoles of M13 target DNA, 10 mg / Contains ml tRNA and 10 ng Pfu FEN-1. All components except enzyme and MgCl ₂ were mixed in a volume of 8 μl and overlaid with 10 μl of CHILLOUT liquid wax. The sample was heated to a reaction temperature of 62 ° C. (This temperature is the optimal temperature at which probe 1 will anneal to the first target). By adding Pfu FEN-1 and MgCl ₂ in 2μl volumes to initiate the reaction. After incubating at 62 ° C. for 15 minutes, the temperature was lowered to 58 ° C. (this temperature is the optimum temperature for Probe 2 to anneal to the second target) and the sample was incubated for an additional 15 minutes. The reaction was stopped with 10 μl of 95% formamide, 20 mM EDTA, 0.02% methyl violet.

  Immediately before electrophoresis, samples of both standard and continuous invasive cleavage reactions were heated at 90 ° C. for 1 minute. The sample was run on a 20% denaturing acrylamide gel (crosslinking 19: 1) containing 7 M urea in 45 mM Tris-borate buffer (pH 8.3) and 1.4 mM EDTA. The gel was then analyzed with Molecular Dynamics FluorImager 595. The resulting image is shown in FIG. 28a. FIG. 28b shows a graph obtained by measuring the fluorescence intensity of each product band.

  In FIG. 28a, lanes 1-5 are generated in a standard invasive cleavage reaction without target (lane 1) and with 10 attomole targets (lanes 2 and 3) or 100 attomole targets (lanes 4 and 5). Product. Uncut probe is observed as a dark band in each lane in the lower half of the panel, and the cleavage product is observed as a smaller black band near the bottom of the panel. The position of the cleavage product is indicated by an arrow on the left side of FIG. The gray ladder bands observed in lanes 1-5 are due to the thermal decomposition of the probe and are not related to the presence or absence of the target DNA. The remaining lanes represent the products produced in a continuous invasive cleavage reaction involving 1 attomole target (lanes 6 and 7), 10 attomole targets (lanes 8 and 9) and 100 attomole targets (lanes 10 and 11). Show. An uncut first probe (probe 1; labeled “1 uncut”) is observed near the top of the panel, and the cut first probe is represented as “1: cut”. Similarly, uncleaved and cleaved second probes are represented as “2: not cleaved” and “2: cleaved”, respectively.

  In the graph of FIG. 28b, the amount of product produced in the standard reaction (“Series 1”) is compared with the amount of product produced in the second step of the continuous reaction (“Series 2”). Background fluorescence levels measured in reactions lacking target DNA were subtracted from each measurement. It can be seen from the table below the graph that the signal of the standard invasive cleavage assay containing 100 attomole of target DNA is almost comparable to the signal of a continuous invasive cleavage assay containing 1 attomole of target. This indicates that inclusion of a second cleavage structure increases the assay sensitivity from 100-fold to 200-fold. The standard assay performed with this enzyme for only 30 minutes does not produce a detectable product in the presence of 10 attomoles of target, but this increase in signal results in subattomoles using a continuous invasive cleavage assay. The target nucleic acid can be easily detected at the level.

  When the amount of target was reduced 10-fold or 100-fold in successive invasive cleavage assays, the signal intensity decreased at the same rate. Even when the reaction is carried out as a series, the quantitative property of the invasive cleavage assay method is retained, and it shows that a nucleic acid detection method having both sensitivity and quantitative property is provided.

  In this example, the two probes used have different optimal hybridization temperatures (ie, experimentally determined temperatures that give the highest turnover at the given reaction conditions), but are identical. It is also possible to select (ie, design) the probe so that it has an optimal hybridization temperature and does not require a temperature shift during incubation.

Example 12
Products after the completion of successive invasive cleavage reactions do not contaminate subsequent similar reactions As described in the "Description of the Invention", a sequence of multiple invasive cleavage events that occur in successive invasive cleavage reactions. In contrast to the repetitive nature of the polymerase chain reaction and similar doubling assays, the product of the first cleavage reaction is not involved in the generation of a new signal in the second cleavage reaction, so a continuous invasive cleavage reaction Means that the product is not contaminated with similar reaction products. When a large amount of termination reactant is added to the newly constructed reaction, the background generated by the amount of brought-in target is combined with the amount of probe already cleaved. In this example, it is shown that a significant portion of the first reaction should be brought into the second reaction to produce a significant signal.

  As described above, the first or first sequential invasive cleavage reaction was performed using 100 attomole of target DNA. A second set of five reactions was prepared by the method described in Example 11 except that a portion of the first reaction was introduced and no other target DNA was included. These second reactions were started and incubated as above. Contains 0%, 0.01%, 0.1%, 1% or 10% of the first reactant. A control reaction containing 100 attomole targets is also included in the second set. By the above method, the reaction was stopped and fractionated by electrophoresis for visualization. The obtained image is shown in FIG. The first probe, the uncut second probe, and the cut second probe are shown on the left as “1: cut”, “2: not cut”, and “2: cut”, respectively.

  In FIG. 29, lane 1 shows the result of the first reaction. The accumulated product is shown at the bottom of the panel. Lane 2 represents a 1:10 dilution of the same reaction to show the level of signal expected from the level of contamination without further amplification. Lanes 3-7 show the results of successive cleavage reactions containing 0%, 10%, 1%, 0.1% or 0.01% of the first reactant, respectively, added as an impurity. Lane 8 also shows a control reaction to which 100 attomole of target DNA was added to confirm the activity of the system in a continuous reaction. When 10% of the pre-cut was transferred (lane 2) and 10% of the target transferred from the lane 1 reaction was further amplified, the signal level in lane 4 was as expected. The signal is not easily distinguished from the background at any level when further diluted. These data are detectable when transferred in large quantities from one reaction to another, but cross-contamination due to small amounts expected from aerosol or equipment contamination does not easily lead to false positive results It is shown that. These data show that when the product of one reaction is deliberately brought into a fresh sample, these products are not involved in the new reaction and do not affect the level of target-dependent signals produced in the reaction. Show.

Example 13
Detection of Human Cytomegalovirus DNA by Invasive Cleavage In the above examples, the ability to detect an invasive cleavage reaction that detects a small amount of viral DNA in the presence of human genomic DNA was demonstrated. In this example, probes and INVADER oligonucleotides that target the region from 3104 to 3061 of the major early gene of human cytomegalovirus (HCMV) shown in FIG. 30 were designed. In FIG. 30, INVADER oligo (89-44; SEQ ID NO: 219) and fluorescein (Fl) labeled probe oligo (89-76; SEQ ID NO: 218) are nucleotides 3057-3110 of viral DNA (SEQ ID NO: 2220). Is shown to anneal along the region of the HCMV genome corresponding to. The probe used in this example is a polypyrimidine probe and, as described herein, the use of a polypyrimidine probe reduces the background signal generated by thermal dissociation of the probe oligo.

Genomic viral DNA was purchased from Advanced Biotechnologies, Incorporated (Columbia, MD). According to Advanced Biotechnologies, the DNA is estimated to be at a concentration of 170 attomole (1 × 10 ⁸ copies) per microliter (but not guaranteed). The same reaction was performed 4 times. Each reaction consists of 1 μM 5 ′ fluorescein labeled probe oligonucleotide 89-76 (SEQ ID NO: 218) in 10 μl of 10 mM MOPS (pH 7.5), 6 mM MgCl ₂ and 0.05% each of Tween20 and Nonidet P-40. One of five for 100 nM INVADER oligonucleotide 89-44 (SEQ ID NO: 219), 1 ng / ml human genomic DNA, and the amount of target HCMV DNA listed above each lane in FIG. And 10 ng of Pfu FEN-1. All components except labeled probe, enzyme and MgCl ₂ were mixed in a final volume of 7 μl and overlaid with 10 μl of CHILLOUT liquid wax. The sample was heated to a reaction temperature of 95 ° C. for 5 minutes and then cooled to 62 ° C. The reaction was started by adding probe, Pfu FEN-1 and MgCl ₂ in a volume of 3 μl. After incubation at 62 ° C. for 60 minutes, the reaction was stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02% methyl violet. Immediately before electrophoresis, samples of both standard and continuous invasive cleavage reactions were heated at 90 ° C. for 1 minute. The sample was run on a 20% denaturing acrylamide gel (crosslinking 19: 1) containing 7 M urea in 45 mM Tris-borate buffer (pH 8.3) and 1.4 mM EDTA. The gel was then analyzed with Molecular Dynamics FluorImager 595.

  The obtained image is shown in FIG. Replication reactions were run in groups of 4 lanes containing the target HCMV DNA content of the reaction shown above each set of lanes (0-170 attomole). Uncut probe (“Uncut 89-76”) is observed in the upper third of the panel, and cleavage product (“Cleaved 89-76”) is observed in the lower third of the panel. It can be seen that the intensity of the accumulated cleavage product is proportional to the amount of target DNA in the reaction. Furthermore, it is clear that the reaction does not contain target DNA that is uncleaved (“no target”) even in the background of human genomic DNA. Each reaction shown in FIG. 31 contains 10 ng of human genomic DNA, but if it contains up to 200 ng of genomic DNA, the amount of accumulated product is slightly affected. 200 ng per 10 μl of reaction mixture was the maximum amount of genomic DNA that was resistant without a significant decrease in signal accumulation. This amount is larger than the DNA in 0.2 ml of urine (usually the amount used for HCMV testing for newborns) and corresponds to the amount found in about 5 μl of whole blood.

These results indicate that the standard (ie non-continuous) invasive cleavage reaction is a sensitive, specific and reproducible tool for the detection of viral DNA. 1.7 Detection of targets attomoles is detected equivalent of roughly 10 ⁶ copies of the virus. This is congenitally equivalent to the number of infecting virus genome in the urine of 0.2mls neonatal (equivalent from 0.2mls per 10 ² to 10 ⁶ genome; Stagno et, J Infect Dis,... 132; 568 [1975]). The use of a continuous invasive cleavage assay allows the detection of fewer numbers of viral DNA molecules and facilitates detection in blood containing higher amounts of heterologous DNA (10 ¹ to 10 ⁵ viruses per ml). Particles; Pector et al., J. Clin. Microbiol., 30: 2359 [1992]).

  From the above, it is clear that the present invention provides reagents and methods that allow the detection and characterization of nucleic acid sequences and mutations in the nucleic acid sequences. The INVADER site-specific cleavage reaction and sequential INVADER site-specific cleavage reaction of the present invention take advantage of the direct detection assay (eg, easy quantification and minimal risk of carry-in contamination) by double or triple oligos. It provides an ideal direct detection method combined with the specificity of nucleotide hybridization assays.

  As described in the “Description of the Invention”, the use of a continuous invasive cleavage reaction can result in low structure remaining, interacting with a second target, and competing with a cleavage probe for binding or produced. There is a problem of the uncut initial or first probe that causes background by level cutting. This is schematically shown in FIGS. 32 and 33. The reaction shown in FIG. 32 utilizes a cleavage product that produces a first cleavage structure that forms an INVADER oligonucleotide for the second cleavage reaction. The structure formed between the second target, the second probe, and the uncut first probe is shown in FIG. 32 as a right-handed structure shown in step 2a. This structure is recognized and cleaved by the 5 ′ nuclease with very low efficiency (ie, less than about 1% under most reaction conditions). However, the resulting product is difficult to distinguish from the specific product and may give false positive results. As described in Example 11, the same effect is obtained when producing an INVADER / target (IT) molecule that incorporates a cleaved first probe. The formation of an unfavorable complex is schematically illustrated in FIG.

  For each of these sequential INVADER assay methods, the improvements made by adding ARRESTOR oligonucleotides at various compositions are shown in the examples below. These ARRESTOR oligonucleotides are designed to bind to residual uncleaved probes from the first cleavage reaction in the series, which increases their potency and reduces non-specific background in subsequent reactions To do.

Example 14
“ARRESTOR” oligonucleotides improve the sensitivity of multiple consecutive invasive cleavage assays This example demonstrates the effect of containing ARRESTOR oligonucleotides on signal generation using IT probe system 33. In the portion that recognizes the target nucleic acid during the first cleavage reaction, the ARRESTOR oligonucleotide hybridizes to the first probe. In addition to investigating the effects of adding ARRESTOR oligonucleotides, the effects of using ARRESTOR oligonucleotides that extend to the region of the first probe that makes up the second IT structure at different distances of complementarity Examined. In order to show non-specific background levels (ie not related to the target nucleic acid) in each set of reaction conditions, these effects can be achieved in reactions that contain target DNA or lack target DNA in a certain concentration range. Compared.

およびリバースプライマー、オリゴ番号：

を用いて実施し、完全長のHBV挿入物である約3.2kbのアンプリコンを増幅した。サイクリングの条件は、95℃5分のプラスミド変性後、95℃30秒-60℃40秒-72℃4分を30サイクルであり、その後最終伸長を72℃10分間実施した。得られたアンプリコンをpAM6#2と命名し、2MのNH₄OAcに調整して、イソプロパノール沈殿で回収した。真空で乾燥した後、DNAを10mMトリス pH.0、0.1mM EDTA中で溶解した。濃度を、既知濃度の標準と比較しながらINVADERアッセイしOD₂₀₀で測定した。 The target DNA for these reactions is a fragment containing the full-length hepatitis B virus genome of a serotype adw strain. This material was prepared from plasmid pAM6 (ATCC # 45020D) using the polymerase chain reaction. PCR is a vector based forward primer, oligo number:

And reverse primer, oligo number:

Was used to amplify an approximately 3.2 kb amplicon, a full length HBV insert. Cycling was performed at 95 ° C. for 5 minutes after plasmid denaturation, followed by 30 cycles of 95 ° C. for 30 seconds-60 ° C. for 40 seconds-72 ° C. for 4 minutes, followed by final extension at 72 ° C. for 10 minutes. The obtained amplicon was named pAM6 # 2, adjusted to 2M NH ₄ OAc, and collected by isopropanol precipitation. After drying in vacuo, the DNA was dissolved in 10 mM Tris pH.0, 0.1 mM EDTA. Concentrations were measured at OD ₂₀₀ by INVADER assay comparing to standards of known concentration.

The INVADER reaction was performed as follows. Five master mixtures named “A”, “B”, “C”, “D” and “E” were prepared. Both mixtures were 12.5 mM MOPS, pH 7.5, 500 femtomolar first INVADER oligo # 218-55-05 (SEQ ID NO: 596), 10 ng human genomic DNA (Novagen) and 30 ng per 8 μl of the mixture. Contains AfuFEN1 enzyme. Mixture A does not contain the amplicon DNA of the additional HBV genome. Mixture B contains 600 molecules of HBV genomic amplicon DNA pAM6 # 2. Mixture C contains 6,000 molecules of pAM6 # 2. Mixture D contains 60,000 molecules of pAM6 # 2. Mixture E contains 600,000 molecules pAM6 # 2. The mixture was dispensed into a reaction tube (8 μl / tube). Mixture A was dispensed into tubes 1, 2, 11, 12, 21, and 22. Mixture B was dispensed into tubes 3, 4, 13, 14, 23 and 24. Mixture C was dispensed into tubes 5, 6, 15, 16, 25 and 26. Mixture D was dispensed into tubes 7, 8, 17, 18, 27 and 28. Mixture E was dispensed into tubes 9, 10, 19, 20, 29 and 30. The sample was incubated at 95 ° C. for 4 minutes to denature the amplicon DNA of the HBV genome. The reaction was then cooled to 67 ° C. and 2 μl of a mixture containing 37.5 mM MgCl ₂ and 2.5 pmoles 218-95-06 (SEQ ID NO: 579) in 2 μl each was added to each sample. The sample was kept at 67 ° C. for 60 minutes. Three kinds of reaction master mixed solutions were prepared. Both mixtures contain 10 pmoles of second probe oligonucleotide # 228-48-04 (SEQ ID NO: 598) in each 2 μl mixture. Mixture 2A contains no other oligonucleotides, Mixture 2B contains 5 pmoles of “ARRESTOR” oligo # 218-95-03 (SEQ ID NO: 599), and Mixture 2C contains 5 pmoles of “ARRESTOR” oligo # 218-95-01 (SEQ ID NO: 600). After incubation at 67 ° C. for 60 minutes (first reaction above), 2 μl of the second reaction mixture was added to each sample. Mixture 2A was added to Sample # 1-10. Mixture 2B was added to Sample # 11-20, and Mixture 2C was added to Sample # 21-30. The temperature was adjusted to 52 ° C and the sample was incubated at 52 ° C for 30 minutes. The reaction was then stopped by adding 10 μl of a solution of 95% formamide, 5 mM EDTA and 0.02% crystal violet. All samples were heated at 95 ° C. for 2 minutes and 4 μl of each sample in 20 mM denaturing acrylamide gel (crosslinked 19) containing 7 M urea in 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA buffer. : Fractionated by electrophoresis in 1). The results were imaged using Molecular Dynamics Fluoroimager 595 with excitation 488 and emission 530. The resulting image is shown in FIG.

  In FIG. 34, Panel A shows the target titration results when no ARRESTOR oligonucleotide is included in the second reaction. Panel B shows the results of a target titration using an ARRESTOR oligonucleotide extended by 2 nucleotides in the non-target complementary region of the first probe. Panel C shows the results of a similar target titration using an ARRESTOR oligonucleotide extended 4 nucleotides to the non-target complementary region of the first probe. The product of the second cleavage reaction is observed as a band near the bottom of each panel. The first two lanes of each panel (ie, 1 and 2, 11 and 12, and 21 and 22) do not contain target DNA, and the signal that migrates with the product band is not affected under each set of conditions. Represents a specific background.

  A visual inspection of these panels shows that both background signals are reduced and that the inclusion of any molecular species of ARRESTOR oligonucleotides increases the predictability. In addition to reducing background in control lanes that do not contain target, reducing background in reactions with more diluted target amounts can give a signal that more accurately reflects the target in the reaction Can thus improve the quantitative range of multiple consecutive invasive cleavage reactions.

  To quantify the impact of including an ARKESTOR oligonucleotide in the second cleavage reaction under these conditions, the average product band signal from the reaction containing the highest amount of target (ie, lanes 9 and 10). Lanes 19 and 20, and the average signal of lanes 29 and 30) compared to the average signal of the control lanes without each panel target, the “fold over background” in each condition set, Determine the signal amplification factor relative to the background. For reactions that do not contain an ARRESTOR oligonucleotide, panel A has a folding background of 5.3, panel B has a folding background of 12.7, and panel C has a folding background of 13.4. If included, the signal specificity is at least doubled relative to the reaction without the ARRESTOR oligonucleotide, and at least in this embodiment of the system, the ARRESTOR oligonucleotide slightly extended toward the non-target complementary region is It is a little more effective. This clearly shows the advantage of using oligonucleotides to increase the specificity of these reactions, particularly those with low levels of target nucleic acids.

Example 15
“ARRESTOR” oligonucleotides can utilize higher concentrations of the first probe without increasing the background signal. By increasing the probe concentration in an invasive cleavage reaction, the amount of given target DNA The amount of gnnal produced can be dramatically increased. Although this explanation is not limited to a specific mechanism, it is believed that increasing the probe concentration increases the apparent rate of turnover of the cleavage reaction by increasing the rate at which the cleaved probe is replaced by the non-cleaved copy. Conceivable. Unfortunately, previously this effect could not be applied to the first cleavage reaction of multiple sequential INVADER assays. This is because the remaining uncleaved first probe can hybridize to the second target and competes with the cleaved molecule, thereby reducing the efficacy of the second reaction. Increasing the concentration of the first probe exacerbates this problem. Furthermore, the complex obtained as described above can be cleaved at a low level, thus contributing to the background. Thus, an increase in the first probe has a double negative effect of slowing the second reaction and increasing the non-target specific background level. This concentration promoting effect can be applied to these second reactions by using ARRESTOR oligonucleotides to sequester or neutralize the remaining first probe.

  To clarify this effect, two types of reactions were performed. In the first reaction, the reaction was performed using a range of concentrations of the first probe, but the ARRESTOR oligonucleotide was not included in the second reaction. In the second reaction, the same concentration of probe was used, but the ARRESTOR oligonucleotide was added to the second reaction.

Each reaction was performed twice. The first INVADER reaction was performed in a final volume of 10 μl, 10 mM MOPS, pH 7.5, 7.5 mM MgCl ₂ , 500 fm first INVADER (218-55-05; SEQ ID NO: 596); 30 ng AfuFEN1 enzyme and 10 ng Of human genomic DNA. All even numbered reactions contain 100 zeptomoles of HBV pAM6 # 2 amplicon (see FIGS. 35A and B). The reaction includes 10 pmoles, 20 pmoles, 50 pmoles, 100 pmoles, or 150 pmoles of the first probe (218-55-02; SEQ ID NO: 601). MOPS, target and INVADER oligonucleotide were mixed to a final volume of 7 μl. The sample was heat denatured at 95 ° C. for 5 minutes and cooled to 67 ° C. During the denaturation of 5 min, it was mixed MgCl _2, probe and enzyme. The first INVADER reaction was initiated by adding 3 μl of MgCl ₂ , probe and enzyme mixture at the final concentrations described above. The reaction solution was kept at 67 ° C. for 30 minutes. The reaction was then cooled to 52 ° C. and the following second reaction components were added to each first INVADER reaction in a total volume of 4 μl. That is, 2.5 pmoles of the second target (Oligo No. 218-95-04; SEQ ID NO: 602); 10 pmoles of the second probe (Oligo No. 228-48-04; SEQ ID NO: 598). Reactions containing ARRESTOR oligonucleotides contained 40, 80, 200, 400 or 600 pmoles of ARRESTOR oligonucleotide (Oligo No. 218-95-01; SEQ ID NO: 600) for each reaction comprising this mixture. A 4-fold molar excess was added relative to the amount of the first probe. The reaction was then incubated at 52 ° C. for 30 minutes. The reaction was stopped by adding 10 μl of a 95% formamide solution containing 10 mM EDTA and 0.02% crystal violet. All samples were heated at 95 ° C. for 1 minute, and 4 μl of each sample was added to a 20% denaturing acrylamide gel containing 7 M urea in a buffer containing 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA ( Fractionation was performed by cross-linking 19: 1) electrophoresis. The results were imaged using a Molecular Dynamics Fluoroimager 595 (excitation 488, emission 530). Images obtained for reactions with or without ARRESTOR oligonucleotides are shown in FIGS. 35A and 35B, respectively. The cleavage product of the second probe is observed as a band near the bottom of each panel.

  In Figure 35A, lane sets 1 and 2 contain 10 pmoles of the first probe, sets 3 and 4 are 20 pmoles, sets 5 and 6 are 50 pmoles, sets 7 and 8 are 100 pmoles, and set 9 and Yo 10 contains 150 picomoles. Increasing the amount of the first probe shows a combined effect that slightly increases the background in lanes that do not contain the target (odd number), but decreases the specific signal in the presence of the target (even numbered lanes), Decreasing the specificity of the reaction, viewed as a measure of “folding background”, indicates that an approach that increases the signal by increasing the probe is impossible to apply to these successive reactions. Can be found by manual search

  In FIG.35B, lane sets 1 and 2 contain 10 pmoles of the first probe, sets 3 and 4 are 20 pmoles, sets 5 and 6 are 50 pmoles, sets 7 and 8 are 100 pmoles, and sets 9 and 10 Contains 150 pmoles. In addition, each reaction contains a 4-fold molar excess of ARRESTOR oligonucleotide added prior to the second cleavage reaction. The background in the target-free lanes (odd) is low in all cases, but the specific signal in the presence of the target (even-numbered lanes) increases with increasing amount of the first probe, It can be seen by visual search that the “folding background” sensitivity at this target level is further increased.

  In order to quantitatively compare these effects, the fluorescence signals of products from non-specific and specific cleavage were measured. The results are shown graphically in FIG. 35C. Shown graphically as a measure of the percentage of the second probe cleaved during the reaction as compared to the amount of first probe used. By examining plots derived from reactions that did not contain the target, the background in the absence of ARRESTOR oligonucleotides was generally about 2-fold higher, both increasing slightly with increasing probe amount. Examined. However, the specific signal is more dramatically different between the two reaction sets. As the first probe increases, the signal of the non-ARRESTOR oligonucleotide reaction steadily decreases, but the signal of the ARRESTOR oligonucleotide reaction continues to increase. At the highest concentration of the first probe tested, the non-ARRESTOR oligonucleotide reaction only has a 1.7-fold specific signal relative to the background, while the ARRESTOR oligonucleotide reaction yields a 6.5-fold signal relative to the background. Having 100 zeptomoles (60,000 copies) of the target is detected, thus improving the continuous invasive cleavage reaction when containing the ARRESTOR oligonucleotide.

Example 16
Skeletal modifications improve the performance of ARRESTOR oligonucleotides, which are all natural “ARRESTOR” oligos that do not contain 3′-amines. The reactions shown in the two examples above use a 2′O-methyl ribose backbone. An ARRESTOR oligonucleotide that was constructed and does not contain a positively charged amine group at the 3 ′ terminal nucleotide was used. Modifications were made to specifically reduce the enzyme that interacts with the first probe / ARRESTOR oligonucleotide complex. It is under development that 2′O-methyl modified oligonucleotides are somewhat resistant to cleavage by 5 ′ nucleases and are slowly degraded by nucleases when using antisense. (See, for example, Kawasaki et al., J. Med. Chem., 36: 831 [1993]).

  When an amino group is present at the 3 ′ end of the oligonucleotide, direct invasive cleavage capacity is reduced. In order to reduce the possibility of the ARRESTOR oligonucleotide forming a cleavage structure in this way, it was designed to include an amino group in the experiments described in this and other examples.

  Originally designed ARRESTOR oligonucleotides (sometimes referred to as “blockers”) do not contain these modifications, and these molecules provide the advantage of reducing the background cleavage of successive invasive cleavage assays I didn't find anything. In fact, it is believed that cleavage may be induced at unexpected sites, possibly contributing to the background, possibly by providing an element for another cleavage structure. The effects of natural and modified ARRESTOR oligonucleotides on background noise in these reactions were investigated in the examples.

Comparison of identical reactions without ARRESTOR oligonucleotides examined the efficacy of all natural ARRESTOR oligonucleotides (ie, ARRESTOR oligonucleotides without base analogs or modifications). Each reaction was performed twice as follows. 2 in each 8 μl containing 12.5 mM MOPS (pH 7.5), 500 femtomolar first INVADER oligonucleotide # 218-55-05 (SEQ ID NO: 596), 10 ng human genomic DNA (Novagen) and 30 ng AfuFEN1 enzyme Different types of master mixes were prepared. Mixture A contains no HBV genomic amplicon DNA, and Mixture B contains 600,000 molecules of HBV genomic amplicon DNA pAM6 # 2. The mixture was dispensed into reaction tubes at 8 μl / tube as follows. Mixture A was dispensed into tubes 1, 2, 5 and 6 and mixture B was dispensed into tubes 3, 4, 7 and 8. The sample was incubated at 95 ° C. for 4 minutes to denature the amplicon DNA of the HBV genome. The reaction was then cooled to 67 ° C. and 2 μl of a mixture containing 37.5 mM MgCl ₂ and 10 pmoles of 218-55-02B (SEQ ID NO: 603) in 2 μl each was added to each sample. The sample was then incubated at 67 ° C. for 30 minutes. Two containing 10 pmoles of second probe oligo # 228-48-04N (SEQ ID NO: 604) and 2.5 pmoles of second target oligonucleotide # 218-95-04 (SEQ ID NO: 602) in 3 μl each A second reaction master mixture was prepared. Mixture 2A contains no other oligonucleotides, and Mixture 2B contains 50 pmoles of natural “ARRESTOR” oligonucleotide # 241-62-02 (SEQ ID NO: 605). After initially incubating at 67 ° C. for 30 minutes, the temperature was adjusted to 52 ° C. and 3 μl of the second reaction mixture was added to each sample as follows. Mixture 2A was added to samples # 1-4. Mixture 2B was added to Samples # 5-8. The sample was then incubated at 52 ° C. for 30 minutes. The reaction was stopped by adding 10 μl of a 95% formamide solution containing 10 mM EDTA and 0.02% crystal violet.

  Both samples were heated at 95 ° C. for 2 minutes, and 4 μl of each sample was added to a 20% denaturing acrylamide gel containing 7 M urea in a buffer containing 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA ( Fractionation was performed by cross-linking 19: 1) electrophoresis. The results were imaged using a Molecular Dynamics Fluoroimager 595 (excitation 488, emission 530). The resulting image is shown in FIG. 36A.

To compare the effects of various modifications to ARRESTOR oligonucleotides, the bases are all natural, but contain an amine at the 3 'end, an ARRESTOR oligonucleotide containing 2'O-methyl nucleotides, and an amine at the 3' end. The reaction was carried out using an ARRESTOR oligonucleotide with a moiety; and an ARRESTOR oligonucleotide that all contained 2′O-methyl nucleotides and an amine at the 3 ′ end. These were compared to reactions without ARRESTOR oligonucleotides. The reaction was carried out as follows. Two kinds of master liquid mixtures were prepared. Both mixes each contain 7 μl of 14.3 mM MOPS (pH 7.5), 500 femtomoles of the first INVADER oligo # 218-55-05 (SEQ ID NO: 596) and 10 ng of human genomic DNA (Novagen). Mixture A contains HBV genomic amplicon DNA, and Mixture B contains 600,00 molecules of HBV genomic amplicon DNA pAM6 # 2. The mixture was dispensed into reaction tubes at 7 μl / tube. Mixture A was dispensed into tubes 1, 2, 5, 6, 9, 10, 13 and 14, and mixture B was dispensed into tubes 3, 4, 7, 8, 11, 12, 15 and 16. The sample was heated at 95 ° C. for 4 minutes to denature the HBV DNA. The reaction was then cooled to 67 ° C. and 3 μl of a mixture containing 25 mM MgCl ₂ , 25 pmoles 218-55-02B (SEQ ID NO: 603) and 30 ng AfuFEN1 enzyme per 3 μl was added to each sample. The sample was then incubated at 67 ° C. for 30 minutes. Four types of reaction master liquid mixtures were prepared. Both mixtures were mixed in 3 μl with 10 pmoles of second probe oligonucleotide # 228-48-04B (SEQ ID NO: 606) and 2.5 pmoles of second target oligonucleotide # 218-95-04 (SEQ ID NO: : 602). Mixture 2A contains no other oligonucleotides, Mixture 2B contains 100 pmoles of natural and amine ARRESTOR oligonucleotide # 241-62-01 (SEQ ID NO: 607), and Mixture 2C contains 100 pmoles of partially O- Oligonucleotide # 241-64-01 (SEQ ID NO: 609) containing oligonucleotide # 241-62-03 (SEQ ID NO: 608) containing methyl and amine, and Mixture 2D is 100 pmol of all O-methyl and amine )including. After first incubating at 67 ° C. for 30 minutes, the temperature was adjusted to 52 ° C. and 3 μl of the second reaction mixture was added to each sample as follows. Mixture 2A was added to samples # 1-4. Mixture 2B was added to Samples # 5-8. Mixture 2C was added to samples # 9-12. Mixture 2D was also added to samples # 13-16. The sample was incubated at 52 ° C. for 30 minutes. The reaction was then stopped by adding 10 μl of a 95% formamide solution containing 10 mM EDTA and 0.2% crystal violet.

  Both samples were heated at 95 ° C. for 2 minutes, and 4 μl of each sample was added to a 20% denaturing acrylamide gel containing 7 M urea in a buffer containing 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA ( Fractionation was performed by cross-linking 19: 1) electrophoresis. The results were imaged using a Molecular Dynamics Fluoroimager 595 (excitation 488, emission 530). The resulting image is shown in FIG. 36B.

  In FIG. 36A, the left panel shows the reaction without the ARRESTOR oligonucleotide and the right panel shows the data for the reaction with the ARRESTOR oligonucleotide that is all natural. The first two lanes in each panel are controls without target and the second set of lanes contains target. The cleavage product is visible in the lower quarter of each panel. The position where the specific reaction product was migrated is indicated by arrows on the left and right.

  Examination of these data revealed that multiple reactions performed in the absence of ARRESTOR oligonucleotide were qualitatively reproducible and significant cleavage occurred only when the target was present. In contrast, when the reaction contained another unmodified oligonucleotide, it was significantly different between multiple lanes (eg, lanes 5 and 6 contained the same reactant but produced significantly different results) ). When all-natural ARRESTOR oligonucleotides were added, the lanes that did not contain these targets would rather have a reduced background and increased cleavage at other sites (i.e. other than those indicated by the arrows across the panel). Band). For this reason, the above modification having the effect shown in FIG. 36B was used.

  The first four lanes in FIG. 36B show the product of the double reaction without the ARRESTOR oligonucleotide and with or without the HBV target (lanes 1 and 2, and lanes 3 and 4, respectively). The next four lanes 5, 6, 7 and 8 used natural ARRESTOR oligonucleotides with a 3 ′ terminal amine. In lanes 9, 10 and 11, 12, ARRESTOR oligonucleotides with a 3 ′ moiety containing 2′O-methyl nucleotides and an amine at the 3 ′ end were used. In lanes 13, 14 and 15, 16 all ARRESTOR oligonucleotides that are 2'O-methyl nucleotides and have a 3 'terminal amine were used. The cleavage product of the second probe is observed in the bottom third of each panel.

  A visual search of these data suppresses the aberrant cleavage seen in Figure 36A by adding an amine to the 3 'end of the native ARRESTOR oligonucleotide, but the ARRESTOR oligonucleotide is a non-ARRESTOR oligonucleotide control. It can be seen that the performance of the reaction is not improved as compared with. In contrast, utilizing 2'O-methyl nucleotides for ARRESTOR oligonucleotides reduces background whether the substitution is partial or complete. In order to quantify the relative effects of such modifications, the fluorescence from each of the simultaneously moving product bands is measured, the signals in the duplicate replication lanes are averaged, and a “folding background” is provided for each reaction containing the target nucleic acid. Calculated.

  If the ARRESTOR oligonucleotide is omitted, the target-specific signal (lanes 3 and 4) is 27-fold over background without target, and 17-fold over background with native ARRESTOR oligonucleotide and amine Gave a signal. Partial 2'O-methyl and amine gave 47-fold signal over background. All 2′O-methyl and amine gave 33 times signal over background.

  These figures show that any modification is effective against the specificity of multiple consecutive invasive cleavage assays. It has also been shown that the use of 2'O-methyl substituted skeletons, either partially or completely, significantly improves the specificity of these reactions. In various embodiments of the invention, whatever the number of modifications, the enhancement is similarly caused by modification of the complex that forms a first target resistant to ARRESTOR oligonucleotides or nucleases.

Example 17
Effect of ARRESTOR oligonucleotide length on signal enhancement in multiple consecutive invasive cleavage assays As described in the "Description of the Invention", the optimal length of an ARRESTOR oligonucleotide depends on the INVADER reaction and other nucleic acid elements. Depends on the design, in particular the design of the first probe. In this example, the effect of the difference in the length of the ARRESTOR oligonucleotide was examined in a system using two different types of second probes. A schematic diagram of these ARRESTOR oligonucleotides that can hybridize to the first probe oligonucleotide in parallel is shown in FIG. 37C. In this figure, the region of the first probe that recognizes the target nucleic acid is underlined. The part that is not underlined and the first base of the part that is underlined is the part that is released by the first cleavage and continues to be involved in the second or subsequent cleavage structure.

Each reaction was performed twice. The INVADER reaction was performed in a final volume of 10 μl, 10 mM MOPS, pH 7.5, mM MgCl ₂ , 500 femtomole of the first INVADER 241-95-01 (SEQ ID NO: 610), 25 pmoles of the first probe 241-95- 02 (SEQ ID NO: 611), 30 ng AfuFEN1 enzyme, and 10 ng human genomic DNA. It may also contain 1 atmol of HBV amplicon pAM 6 # 2. MOPS, target DNA, and INVADER oligonucleotide were mixed to a final volume of 7 μl. The sample was heat denatured at 95 ° C. for 5 minutes and cooled to 67 ° C. During the denaturation of 5 min, it was mixed MgCl _2, probe and enzyme. The first INVADER reaction was initiated by adding 3 μl of MgCl ₂ , probe, and enzyme mixture at the above final concentrations. The reaction solution was kept at 67 ° C. for 30 minutes. The reaction was then cooled to 52 ° C. and the following second reaction components were added to each first INVADER reaction in a total volume of 3 μl. That is, 2.5 pmoles of the second target 241-95-07 (SEQ ID NO: 612); 10 pmoles of the second probe 228-48-04 (SEQ ID NO: 598) or 228-48-04N (SEQ ID NO: 604) ) And 241-95-03 (SEQ ID NO: 613), 241-95-04 (SEQ ID NO: 614), 241-95-05 (SEQ ID NO: 615) or 241-95-06 (SEQ ID NO: 616) Any 100 picomolar ARRESTOR oligonucleotide. As a control for the effect of the ARRESTOR oligonucleotide, a reaction in which the ARRESTOR oligonucleotide was omitted was used.

  The reaction was incubated at 52 ° C. for 34 minutes, then the reaction was stopped by adding 10 μl of a 95% formamide solution containing 10 mM EDTA and 0.02% crystal violet. All samples were heated at 95 ° C. for 1 minute, and 4 μl of each sample was added to a 20% denaturing acrylamide gel containing 7 M urea in a buffer containing 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA ( Fractionation was performed by cross-linking 19: 1) electrophoresis. The results were imaged using a Molecular Dynamics Fluoroimager 595 (excitation 488, emission 530). Images obtained for reactions involving shorter and longer second probes are shown in FIGS. 37A and 37B, respectively.

  In each figure, the cleavage product is observed as a band in the lower half of each lane. The first 4 lanes in each figure do not contain the ARRESTOR oligonucleotide and are the products of the double reaction with or without the HBV target (lane sets 1 and 2 respectively), and in the next 4 lane sets 3 and 4 The shortest ARRESTOR 241-95-03 (SEQ ID NO: 613) was used. In lanes 5 and 6, 241-95-04 (SEQ ID NO: 614) was used. In lanes 7 and 8, 241-95-05 (SEQ ID NO: 615) was used. In lanes 9 and 10, 241-95-06 (SEQ ID NO: 616) was used.

  The main background in question is the “target free” control lane (odd; this band migrates with the target specific signal near the bottom of each gel panel). It can be seen that the shortest ARRESTOR oligonucleotide is least effective in suppressing this background and becomes more potent when the ARRESTOR oligonucleotide is further extended to the part involved in subsequent cleavage reactions. Even with this difference in effect, these data show that the design tolerance of ARRESTOR oligonucleotides is broad. The choice of length depends on the temperature at which the reaction with the ARRESTOR oligonucleotide is performed, the duplex formed between the first probe and the target, and between the first probe and the second target. It is affected by the length and the relative concentration of different nucleic acid species in the reaction.

Example 18
Effect of ARRESTOR oligonucleotide concentration on signal enhancement in multiple sequential invasive cleavage assays When investigating the effects of including ARRESTOR oligonucleotides in these cleavage reactions, the first probe is at an excessive concentration. The focus was to determine whether the concentration of the ARRESTOR oligonucleotide affected the yield of non-specific or specific signals and whether the length of the ARRESTOR oligonucleotide was a factor. These two variables were examined in the following examples.

Each reaction was performed twice. The first INVADER reaction was performed in a final volume of 10 μl, 10 mM MOPS, pH 7.5, 7.5 mM MgCl ₂ , 500 femtomole of the first INVADER 241-95-01 (SEQ ID NO: 610), 25 pmol of the first probe 241-95-02 (SEQ ID NO: 611); contains 30 ng AfuFEN1 enzyme and 10 ng human genomic DNA. When the target DNA is included, 1 attomole of the above HBV amplicon pAM 6 # 2 is included. MOPS, target and INVADER oligonucleotide were mixed to a final volume of 7 μl. The sample was heat denatured at 95 ° C. for 5 minutes and cooled to 67 ° C. During the denaturation of 5 min, it was mixed MgCl _2, probe and enzyme. The first INVADER reaction was initiated by adding 3 μl of MgCl ₂ , probe and enzyme mixture at the final concentrations described above. The reaction solution was kept at 67 ° C. for 30 minutes. The reaction was then cooled to 52 ° C. and the following second reaction components were added to each first INVADER reaction. That is, 2.5 pmoles of the second target 241-95-07 (SEQ ID NO: 612), 10 pmoles of the second probe 228-48-04 (SEQ ID NO: 598), and 50, 100, or 200 pmoles of May also contain ARRESTOR oligonucleotides 241-95-03 (SEQ ID NO: 613) or 241-95-05 (SEQ ID NO: 615). The total volume was 3 μl. The reaction was then incubated at 52 ° C. for 35 minutes. The reaction was stopped by adding 10 μl of a 95% formamide solution containing 10 mM EDTA and 0.02% crystal violet. All samples were heated at 95 ° C. for 1 minute, and 4 μl of each sample was added to a 20% denaturing acrylamide gel containing 7 M urea in a buffer containing 45 mM Tris-borate (pH 8.3) and 1.4 mM EDTA ( Fractionation was performed by cross-linking 19: 1) electrophoresis. The results were imaged using a Molecular Dynamics Fluoroimager 595 (excitation 488, emission 530). The obtained image is shown in FIG. 38 as a composite image.

  Each duplicate reaction was added to adjacent lanes in the gel to give a single lane number. All odd numbered lanes are controls that do not contain target. Lanes 1 and 2 do not contain the ARRESTOR oligonucleotide. Lanes 3-8 show the results of reactions containing shorter ARRESTOR oligonucleotides 241-95-03 (SEQ ID NO: 613). Lanes 9-14 show the results of the reaction containing the longer ARRESTOR oligonucleotide 241-95-05 (SEQ ID NO: 615). The cleavage product of the second reaction can be observed in the lower third of each panel. A visual search of these data (ie, comparison of specific products to background bands) shows that each ARRESTOR oligonucleotide is effective at any concentration.

  To quantify the relative effects of ARRESTOR oligonucleotide length and concentration, each of the simultaneously moving product bands was measured, the double lane signal was averaged, and the “folded background” (signal + target / signal− Target) was calculated for each reaction containing the target nucleic acid. The reaction without the ARRESTOR oligonucleotide gave a signal approximately 27 times the background. Inclusion of 50, 100 or 200 pmoles of shorter ARRESTOR oligonucleotides produced 42-fold, 51-fold and 60-fold products, respectively, relative to the background. This indicates that the lowest concentration of short ARRESTOR is less effective than longer ARRESTOR oligonucleotides (see, eg, the examples above), and by increasing the concentration of the ARRESTOR oligonucleotide, the ARRESTOR oligonucleotide : The first probe ratio can be increased and compensated.

  In contrast, containing longer ARRESTOR oligonucleotides at 50, 100, or 200 pmol produced 60-, 32-, and 24-fold products, respectively, relative to the background. The effect of this longer ARRESTOR oligonucleotide when compared to the shorter ARRESTOR oligonucleotide at the lowest concentration was consistent with the above example. However, as the concentration increased, the yield of specific product decreased. This suggests an effect competing with the elements of the second cleavage reaction.

  This data shows that ARRESTOR oligonucleotides can be used favorably in the design of multiple specific reactions. The choice of concentration depends on the temperature at which the reaction with the ARRESTOR oligonucleotide is performed, between the first probe and target, and between the first probe and second target, and between the first probe and ARRESTOR. It is affected by the length of the duplex formed between the oligonucleotides.

  Molecular biology for the selection of target nucleic acid oligonucleotides other than HBV as described herein (eg, oligonucleotide composition and length) and optimization of cleavage reaction conditions according to the model described herein. The usual methods known to those skilled in the art and the usual practices are followed.

  Example 10 shows that there are also enzymes that require overlap between the upstream INVADER oligonucleotide and the downstream probe oligonucleotide to create a cleavage structure (FIG. 27). Even if it is only an overlapping base in the INVADER oligonucleotide (eg, with the HCMV probe shown in FIG. 30), the nucleotide at the 3 ′ end of the INVADER oligonucleotide need not be complementary to the target strand. It became clear. The requirement for duplication can provide a basis for simplicity in detecting single nucleotide polymorphisms (SNPs) or mutations in nucleic acid samples.

  To detect single base mutations, at least two oligonucleotides (eg, probes and INVADER oligonucleotides) are hybridized in tandem to the target nucleic acid, resulting in an overlapping structure recognized by the CLEAVASE enzyme in the reaction. Form. Unpaired “flaps” are included at the 5 ′ end of the probe. The enzyme recognizes the overlap and cleaves the unmatched flap and releases it as a target-specific product. Enzymes with strong selectivity for overlapping structures, ie enzymes that cleave overlapping structures at a much higher frequency than cleaving non-overlapping structures, include FEN from Archaeobus fulgidus and Pyrococcus furiosus -1 enzymes, such enzymes are particularly preferred for the detection of mutations and SNPs.

Example 19
Kits for Performing an mRNA INVADER Assay In certain embodiments, the present invention provides kits that include one or more of the components necessary to practice the present invention. For example, the present invention provides kits for storing or delivering the enzymes and / or reaction components (eg, INVADER assay) of the present invention necessary to perform a cleavage assay. As an example, however, without limiting the kit of the present invention to a specific arrangement or combination of components, one embodiment of a kit for carrying out the present invention is described in the following section.

In certain embodiments, the kits of the invention provide the following reagents:

  FIG. 41 shows examples of the first oligonucleotide and the second oligonucleotide that are preferably used in the method of the present invention. While the indicated oligonucleotides are utilized in several methods and variations of the invention, these INVADER assay oligonucleotide sets have particular application in the kits of the invention. The oligonucleotide sets shown in FIG. 41 can be used as individual sets to detect individual target RNAs. Alternatively, design of these probe sets (eg, oligonucleotides and / or sequences thereof) that can be combined in double or multiple reactions to detect two or more analytes or controls in a single reaction May be utilized for DNA detection assay applications using the guidelines for reaction design and optimization provided herein. Another oligonucleotide utilized in the detection assays and kits of the invention is shown in FIG.

In certain embodiments, the kits of the invention provide a list of additional elements (eg, reagents, providers, and / or equipment) that are supplied by the user to perform the methods of the invention. For example, the list of such additional elements is not limited to a specific element, but one example of such a list includes the following.
・ RNase-free H ₂ O (eg, DEPC-treated)
Transparent CHILLOUT-14 liquid wax (MJ Research) or RNase-free optical grade mineral oil (Sigma, catalog number M-5904)
・ Phosphate buffered saline (without MgCl ₂ and CaCl ₂ )
・ 96-well polypropylene microplate (MJ Research, Catalog No. MSP-9601)
・ 0.2ml thin wall tube ・ Thermaseal well tape (eg GeneMate, catalog number T-2417-5)
・ Multi-channel pipette (0.5-10μl, 2.5-20μl, 20-200μl)
Thermal cycler or other heating source (eg laboratory dryer or heating block)
・ Fluorescence microplate reader (Preferred plate reader is a top-reading type equipped with an optical filter and has the following properties)

In some embodiments, the kits of the invention provide a list of optional elements (eg, reagents, providers, and / or equipment) that are supplied by the user to facilitate the performance of the methods of the invention. . For example, although such a list of selective elements is not limited to a specific element, one aspect of such a list is as follows.
・ TRNA solution, 20ng / μl (Sigma, R-5636)
・ 1x stop solution (10 mM Tris-HCl, pH 8, 10 mM EDTA)
• Black opaque 96-well microplate (eg, COSTAR, catalog number 3915)
・ Electric reversible pipette (250μl)

  In some embodiments of the kit, detailed protocols are provided. In preferred embodiments, INVADER assay reaction preparation protocols (eg, formulations, preferred methods of making reaction mixtures) are provided. In a particularly preferred embodiment, the reaction mixture preparation protocol includes computer or graph assistance to reduce the risk of failure in carrying out the methods of the invention (eg, calculating the volume of reagents required for multiple reactions). Tables to facilitate, and guide plate layout to assist in the placement of multi-well assay plates containing multiple assay reactions). Although such protocols are not limited to specific components or forms, by way of example, the kit of the present invention includes the following protocols.

I. Detailed protocol for mRNA INVADER assay
1. Develop a microplate layout plan for each experimental run. An example of a control microplate layout with 40 samples, 6 standards, and no target is shown in FIG. A target-free control composition (tRNA carrier or 1x cell lysis buffer 1) and standard for quantification are required for absolute quantification.

  2. Prepare first reaction mixture for single or dual assay. To calculate the volume of reaction composition required for the assay (X volume), multiply the number of reactions (both sample and control) by 1.25 [x volume (μl) = number of reactions x 1.25]. After adding the last reagent, vortex the first reaction mixture briefly and mix well. Dispense 5 μl per microplate from the first reaction mixture (in this step, an electric repeat pipette is recommended).

二重アッセイ・フォーマット

First reaction mixture Single assay format

Dual assay format

  3. Add 5 μl each of control, standard or sample (total RNA or cell lysate) without target to the appropriate wells and mix by pipetting 1-2 times. Overlay each reaction with 10 μl of clear CHTLLOUT or mineral oil. Seal the microplate with therma seal well tape.

  4. Incubate the reaction at 90 ° C for 60 minutes in a thermal cycler or dryer.

  5. Prepare a second FRET reaction mixture for single or dual assays while keeping the first reaction warm. Multiply the number of reactions (both sample and control) by 1.25 [X volume (μl) = number of reactions × 1.25] to calculate the required composition volume (X volume). Add a portion of the second FRET reaction to multiple 0.2 ml thin-walled tubes or 8 strips (70 μl / tube is sufficient for 12 reaction rows).

二重アッセイ・フォーマット

Second FRET reaction mixture, single assay format

Dual assay format

  6. After the incubation of the first reaction is complete, unseal the microplate and add 5 μl of the second FRET reaction mixture per well using a multichannel pipette. Mix by pipetting 1-2 times. Reseal the microplate with well tape and incubate the microplate at 60 ° C. for 60 or 90 minutes as indicated on the product information sheet. The incubation time for the second reaction may be different. For details, see Section 2 of “Notes on mRNA INVADER Assay Procedures”.

Responses can be measured using any of the 7.2 methods ( direct measurement or stop and transfer ).
Note: Remove the microplate seal before performing the microplate measurement.

注意：至適ゲインの設定は装置毎に異なるので、最良のシグナル／バックグラウンド比（標的を含まない対照のシグナルで除した試料の生のシグナル）、または標的を含まない対照試料の測定値（約100RFU）を与えるようにゲインを調整する。光源としてキセノンランプを使用する蛍光マイクロプレート読み取り装置は一般的に、より高いRFUを与える。マイクロプレートの直接測定については、製造元の推奨に従い調整を行うために、蛍光マイクロプレート読み取り装置の探針の高さ、およびプレートがどのように配置されているかを知る必要がある。 Direct measurement method. In this method, multiple data sets can be acquired to expand the dynamic range of the assay. During the second INVADER reaction, the microplate can be measured directly with a top-reading fluorescent microplate reader.
The recommended settings for the PerSeptive Biosystem Cytofluor 4000 instrument are shown below.

Note: Since the optimal gain setting varies from instrument to instrument, the best signal / background ratio (the raw signal of the sample divided by the control signal without the target) or the measurement of the control sample without the target ( Adjust the gain to give about 100RFU. Fluorescent microplate readers that use a xenon lamp as the light source generally give higher RFU. For direct measurement of the microplate, it is necessary to know the height of the probe of the fluorescent microplate reader and how the plates are arranged in order to make adjustments according to the manufacturer's recommendations.

Stop and transfer method
1. Prepare IX stop solution (10 mM Tris-HCl (pH 8), 10 mM EDTA) containing RNase-free H ₂ O. Using a multichannel pipette, add 100 μl per well.
2. Transfer 100 μl of diluted reaction to a black microplate (eg, COSTAR (Corning), catalog number 3915).
3. Measure the microplate using the same parameters as the direct measurement method, but adjust the gain so that the measured value of the target control sample is about 100 RFU (see “Caution” above). deep.

  In certain embodiments, supplemental documentation is provided, such as protocols for auxiliary methods, eg, additional reagents, or auxiliary methods for sample preparation for use in the methods of the invention. In a preferred embodiment, the supplemental documentation includes a list of guidelines and precautions so that even unskilled or inexperienced users can use the method and kit. In a particularly preferred embodiment, the supplementary document contains problem-solving guidelines, such as guidelines that describe problems that users may encounter, and suggestions for solutions or corrections that assist the user in solving or avoiding such problems. Including guidelines to give

  Although such supplementary documents are not limited to specific contents, for example, the kit of the present invention includes the following methods and guidelines.

II. Preventing RNase Contamination To prevent RNase contamination during sample preparation and testing, in one aspect, inform users to pay attention to the following precautions.
• Always wear disposable gloves to avoid contact with samples and reagents.
• To prevent cross-contamination, sample and assay reagent preparation uses a guaranteed RNase-free disposable product that includes a thin-walled polypropylene tube and a pipette tip with an aerosol barrier.
• Use RNase-free (DEPC-treated) H ₂ O for sample and / or reagent dilution.
• Keep RNA samples and controls on ice during assay preparation.

III. Sample and control preparation Note: Dilute both standards and samples to a concentration corresponding to the addition of 5 μl per reaction.
Example 1: The standard concentration of 5 attomoles is 1 attomole / μl. 1 atmol = 10 ^-18 = 602,000 molecules.
Example 2: The concentration of a 100 ng sample is 20 ng / μl.

A. Control preparation
Control without target:
Total RNA format: tRNA carrier (20ng / μl)
Cell lysate format: 1x cell lysis buffer 1 (10x cell lysis buffer 1 is diluted with 1xRNase-free H ₂ O)
Positive control:
RNA standard (Std) (100 atmol / μl in vitro transcript)
1. Positive control is tRNA carrier (when using total RNA sample) or 1x cell lysis buffer 1 [10x cell lysis buffer 1 diluted with 1xRNase-free H ₂ O] (use cell lysate sample Prepare the RNA standard by diluting with The product information sheets included in each kit show recommended standard test concentrations and preparation methods.
2. It is recommended to use a new set of standards in each implementation. Keep standards on ice while preparing for reaction.

B. Preparation of total RNA sample
1. Total RNA is prepared from cells or tissues according to the manufacturer's instructions for the selected preparation method. Recommended methods include TRIZOL (Life Technologies, Rockville, MD), RNEASY (Qiagen, Valencia, CA) and RNA WIZ (Ambion, Austin, TX).
2. Dilute the total RNA sample to an appropriate concentration with RNase-free H ₂ O.

C. Preparation of cell lysate sample-Using a 96-well microplate Note: The detection method using this cell lysate uses adherent cells cultured in a 96-well tissue culture microplate. Usually, 10,000 to 40,000 cells are seeded per well. Depending on the cell type and / or mRNA expression level, different seeding densities may be required. See "Procedure Notes" for more details. For cells that show high expression, the following method can be used to attenuate the signal of cell lysate:
• Seed with a small number of cells per well.
• Dilute cell lysate with 1x cell lysis buffer 1 (eg 2.5 μl lysate + 2.5 μl 1x cell lysis buffer 1) before adding to reaction.
• After adding the second FRET reaction mixture, measure the microplate reaction for 15-30 minutes instead of the recommended 60-90 minutes.
1. Dilute 10x cell lysis buffer 1 to 1x concentration with RNase-free H ₂ O.
2. Using a multichannel pipette, carefully remove the media from the wells of adherent cells without disturbing the monolayer cells.
3. Wash the cells once with 200 μl PBS (without MgCl ₂ and CaCl ₂ ) and carefully remove the remaining PBS with a multichannel pipette.
4. Add 40 μl of 1 × cell lysis buffer 1 per well. Lyse cells for 3-5 minutes at room temperature.
5. Using a multichannel pipette, carefully transfer 25 μl of each lysate sample to a 96-well microplate. Avoid transferring cell samples from the bottom of the well.
6. Overlay each lysate sample with 10 μl of clear CHILLOUT or mineral oil (no overlay is required if using a thermal cycler with a heatable lid).
7. Seal the microplate with therma seal well tape. Immediately heat the lysate for 15 minutes at 75-80 ° C. in a thermal cycler or dryer to inactivate cellular nucleases.
8. Proceed with the reaction preparation during the warming process. See the detailed protocol for mRNA INVADER assay (above) for explanation.
9. Immediately after the heat inactivation step, add the lysate sample to the reaction microplate. Alternatively, the lysate sample may be rapidly transferred to a -70 ° C freezer for later testing (long-term stability is not clear and depends on each cell type).

IV. Attention assay method for mRNA INVADER procedure
1. Optimization of RNA sample type and RNA sample volume Optimize assay performance using total RNA samples prepared from tissues or cells. Confirmation of multiple total RNA preparation methods / kits for the performance of the mRNA INVADER assay.
・ TRIZOL (Life Technologies, Rockville, MD)
・ RNeasy (Qiagen, Valencia, CA)
・ RNA WIZ (Ambion, Austin, TX)

  It is important to use methods or kits that minimize the level of genomic DNA that can inhibit signal generation. Preliminary experiments are recommended to determine the amount of total RNA sample that gives the maximum detection limit and dynamic range (usually 1 to 200 ng, depending on the expression level of the gene).

  In addition, assay methods were confirmed using lysate samples of a plurality of cell types. The recommended cell density in 96-well tissue culture microplates is 10,000-40,000 cells per well, but depends on the cell type and expression level of the gene of interest. It is recommended that preliminary experiments be performed on a given cell line and / or gene to be monitored. Such experiments include diluting the lysate sample with different levels of cell density and / or 1x cell lysis buffer 1 (eg, when adding 5 μl of sample and 1 μl of lysate sample and Prepare a test level of 1 μl by mixing 4 μl of 1 × cell lysis buffer 1).

2. Adjusting the dynamic range: changing the incubation time of the second reaction For most specimens, the incubation time for the second reaction is sufficient for the time indicated in the protocol. However, the linear detection range (signal / background <15-25) can be adjusted by varying the time after addition of the second FRET reagent and measuring the reaction microplate. For example, it is often detected in 15 to 30 minutes in a highly expressed sample. In the direct measurement method (step 7 of the detailed protocol of the mRNA INVADER assay), if the test sample does not give sufficient signal due to the early measurement, the reaction microplate can be further incubated, so the second The reaction time can be easily optimized.

  By tracking the time course of the fluorescence signal of the second reaction, the dynamic range of the assay can also be expanded. Direct measurement at multiple time points enables the use of low cost devices. Alternatively, a dynamic range comparable to that of other mRNA quantification methods can be achieved using a real-time fluorescence device.

3. Dynamic range adjustment: changing the sample level
While the FRET detection method greatly simplifies the assay, the dynamic range is usually limited to a few on a logarithmic scale when endpoint measurement is used. However, because the mRNA INVADER assay signal is generated linearly with respect to both target level and time, it can be used to extend the dynamic range beyond the logarithmic scale of 3 (eg, required for highly expressed genes). The simplest is to adjust the level of the total RNA sample. Using the normalized sample signal, the change in gene expression ratio (the signal of the treated sample is divided by the signal of the untreated sample) can be calculated with high reliability. This can be achieved by testing the level of the sample giving a signal within the linear detection range determined by the standard curve. For example, the induction ratio of a sample induced to a high level is calculated as follows.
Induction ratio = (net signal of 1 ng treated sample x 100) / net signal of 100 ng untreated sample

V. Problem-solving guidelines

Appendix A
mRNA INVADER Single Assay Worksheet
Prepare mRNA INVADER assay , samples and controls.
Prepare the first reaction mixture. Vortex briefly and dispense 5 μl per well.
Add 5 μl of sample or control per well and pipette 1 to 2 times.
Add 10 μl CHILLOUT or mineral oil per well.
Incubate the first reaction at 60 ° C for 90 minutes.
• Prepare a second FRET reaction mixture and vortex briefly.
• Dispense 5 μl per well using a multichannel pipette and pipette 1-2 times.
Incubate the second reaction at 60 ° C for 90 minutes.
• Measure the microplate with a fluorescent microplate reader (FAM dye: Ex. 485 nm / Em. 530 mn).

第二のFRET反応混合液

First reaction mixture

Second FRET reaction mixture

Appendix B
mRNA INVADER Duplex Assay Worksheet
Prepare mRNA INVADER assay , samples and controls.
Prepare the first reaction mixture. Dispense 5 μl per well.
Add 5 μl of sample or control per well and pipette 1 to 2 times.
Add 10 μl CHILLOUT or mineral oil per well.
Incubate the first reaction at 60 ° C for 90 minutes.
• Prepare a second FRET reaction mixture and vortex briefly.
• Dispense 5 μl per well using a multichannel pipette and pipette 1-2 times.
Incubate the second reaction at 60 ° C for 90 minutes.
Measure the microplate with a fluorescent microplate reader (FAM dye: Ex. 485 nm / Em. 530 nm; red dye: Ex. 560 nm / Em. 620 nm).

第二のFRET反応混合液

First reaction mixture

Second FRET reaction mixture

(Table 2)

B．RNA活性表

(Table 3) Planned mutations in region A of the polymerase
A. DNA activity table

B. RNA activity table

RNA活性表

(Table 4) Planned arch mutation
DNA activity table

RNA activity table

RNA活性表

(Table 5) Arch / Sum combination
DNA activity table

RNA activity table

RNA活性表

(Table 6) Random mutagenesis of helix-hairpin-helix
DNA activity table

RNA activity table

RNA活性表

(Table 7) Random thumb mutation
DNA activity table

RNA activity table

B．RNA活性表

(Table 8) Chimeric mutants
A. DNA activity table

B. RNA activity table

  All publications and patents mentioned above in this specification are herein incorporated by reference. It will be appreciated by those skilled in the art that various modifications and variations can be made from the described method and system of the present invention without departing from the scope and spirit of the invention. Although the invention has been described in connection with particularly preferred embodiments, it is not appropriate to limit the claims of the invention to only such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the claims.

Claims (14)

  A polypeptide comprising the amino acid sequence of SEQ ID NO: 37. SEQ ID NO: 35, 36 and polypeptide comprising an amino acid sequence selected from the group consisting of 52. A nucleic acid encoding the polypeptide according to claim 1 or 2 . 4. The nucleic acid of claim 3 , wherein the nucleic acid is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 104. 4. The nucleic acid according to claim 3 , wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 102, 103, and 119. An expression vector comprising the nucleic acid according to any one of claims 3 to 5 . A host cell comprising the expression vector according to claim 6 . A kit comprising the polypeptide according to claim 1 or 2 . 9. The kit of claim 8 , further comprising at least one nucleic acid cleavage substrate comprising a DNA portion and at least one RNA component. The kit according to claim 8 or 9 , further comprising a labeled oligonucleotide. A method for cleaving nucleic acids, comprising:
a) i) the polypeptide of claim 1 or 2 , and
ii) providing a sample comprising a substrate nucleic acid, and
b) A method comprising exposing the substrate nucleic acid to the polypeptide. 12. The method of claim 11 , wherein exposure of the substrate nucleic acid to the polypeptide produces at least one cleavage product. 13. The method of claim 12 , further comprising c) detecting the cleavage product. 14. The method according to any one of claims 11 to 13 , wherein the sample containing the substrate nucleic acid contains a cell lysate.

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