JP2008029335A - Primer set and kit for use in new gene amplification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new nucleic acid amplification method to which the reaction principle of the LAMP(loop-mediated isothermal amplification) method is applied. <P>SOLUTION: A primer set for amplifying a target nucleic acid sequence is provided, being characterized in containing at least two primers(A) each comprising a nucleic acid sequence region(a), a nucleic acid sequence region (b) complementary to the sequence of the region (a), and a nucleic acid sequence region (c) complementary to a region(c') in a target nucleic acid sequence, in this order from the 3' terminal side to the 5' terminal side of the primer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a primer set and kit used for a novel gene amplification method.

  Gene mutations may cause missense mutations with changes in translated amino acids, silent mutations without amino acid changes, and frame shift mutations that shift the translation frame due to base deletions or insertions. In addition, gene mutations may lead to gene translational abnormalities through splicing abnormalities and the like, and mutations other than silent mutations often involve structural or functional changes in proteins. Furthermore, the mutation in the protein expression control region may affect the protein expression control mechanism.

  Among the differences in the nucleotide sequences of nucleic acids, a mutation present at a frequency of 1% or more in a certain population is particularly called a polymorphism. Here, the group means a group distinguished by geographical isolation or subspecies. Among polymorphisms, polymorphisms due to single nucleotide insertions, deletions, and substitutions are particularly called single nucleotide polymorphisms (hereinafter abbreviated as SNPs). SNPs are attracting attention because they are the most frequently occurring polymorphisms in the human genome. Since polymorphism spreads to the population at a certain frequency, it is considered that it does not change any traits at all, or influences traits that can be said to be constitutions, not traits that are particularly disadvantageous to survival or reproduction. It is done.

  For polymorphisms, mutations found in less than 1% are mutations that do not spread within the population in humans and are most likely related to disease. That is, it corresponds to a mutation found in a genetic disease. In addition, some mutations found in individuals are related to diseases like mutations found in cancer and the like. Detection of such mutations provides definitive information in the diagnosis of the corresponding disease.

  Whether or not the base sequence of a gene is different from the predicted base sequence can be confirmed by hybridization of complementary base sequences. Specifically, hybridization of primers and probes is used. For example, a primer for nucleic acid amplification can act as a primer only when the target base sequence has a base sequence complementary to the primer. Based on this principle, it can be determined whether or not the target base sequence is complementary to the primer using the nucleic acid amplification product as an index.

  A PCR (Polymerase Chain Reaction) method is known as one of nucleic acid amplification methods (Non-patent Document 1). However, the method for confirming a base sequence based on PCR has several problems. In PCR, if complementary strand synthesis is performed by mistake, the product functions as a template for the subsequent reaction and causes an incorrect result. Furthermore, in the PCR method, a special temperature control device is necessary for the implementation, the amplification reaction proceeds exponentially, there is a problem in quantitativeness, the sample and the reaction solution are contaminated from the outside, There are problems such as being easily affected by contamination in which nucleic acid mistakenly mixed functions as a template.

  As a nucleic acid amplification method that can be carried out under isothermal conditions and can maintain the reaction specificity at a high level compared to PCR, a LAMP (Loop-Mediated Isolation Amplification) method is used (Patent Literature). 1, Non-Patent Document 2). In addition, a method for detecting SNPs by applying the LAMP method (Non-patent Document 3) and a nucleic acid amplification method by improving the LAMP method (Patent Document 2) have also been reported. The LAMP method is characterized in that the nucleic acid amplification reaction is significantly inhibited when the base sequence of the template is different from the design. Due to this feature, high specificity is realized by a mutation detection method based on the LAMP method.

International Publication No. 00/28082 Pamphlet International Publication No. 2002/090538 Pamphlet Science, 230, 1350-1354, 1985 T.A. Notomi et al. Nucleic Acid Res. , 2000, Vol. 28, no. 12, e63 The 3rd International Works on Advanced Genomics 2000.11.13-14; Yokohama, A Novel SNP Typology Technology on MPA. Eiken Chemical Co., Ltd., “Principle of LAMP Method” (Primer Design), 2005, Internet <URL: http: // looppamp. eiken. co. jp / lamp / primer. html>

  However, the LAMP method and its improved method have a problem that a special primer structure is required and the degree of freedom in primer design is limited. For example, as shown in FIG. 1, the primer used in the LAMP method needs to have a sequence complementary to the template nucleic acid over two regions. Furthermore, it is necessary to optimize the inter-primer distance, the melting temperature of each primer (hereinafter also referred to as Tm value), the terminal stability of each primer region, and the GC content. There is a limitation in primer design such that the end must not be complementary (see Non-Patent Document 4).

  An object of the present invention is to provide a new nucleic acid amplification method that applies the reaction principle of the LAMP method and solves the above-described problems.

  As a result of repeated studies to solve the above problems, the present inventors have found that nucleic acid amplification can be performed on a new principle by applying a primer set having a specific structure to the LAMP method. completed. That is, this invention consists of the following structures.

<1> From the 5 ′ end to the 3 ′ end, the nucleic acid sequence region (a), the nucleic acid sequence region (b) complementary to the sequence of the region (a), and the region (c ′) in the target nucleic acid sequence A primer set for amplifying a target nucleic acid sequence, comprising at least two kinds of primers (A) containing complementary nucleic acid sequence regions (c).
<2> In addition to the primer (A), it comprises at least two kinds of primers (B) having a nucleic acid sequence region (d) complementary to the region (d ′) in the target nucleic acid sequence, <1> (However, in the target nucleic acid sequence, the region (d ′) is present on the 3 ′ end side of the region (c ′)).
<3> The primer set according to <1> or <2> for use in amplification of a target nucleic acid sequence under isothermal conditions.
<4> The primer set according to any one of <1> to <3>, wherein each of the regions (a) and (b) comprises 50 bases or less.
<5> The sequence existing between the regions (a) and (b) consists of 10 bases or less, or the regions (a) and (b) are adjacent to each other, <1> ~ Primer set according to any one of <4>.
<6> The primer set according to any one of <1> to <5>, wherein the regions (a) and (b) are composed of only adenine (A) and thymine (T). .
<7> The primer set according to any one of <1> to <6>, wherein the Tm value of the region (c) is higher than the Tm value of the region (a).
<8> Nucleic acid amplification in which a target nucleic acid sequence in a nucleic acid sample is amplified in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set according to any one of <1> to <7> are present Method.
<9> The nucleic acid amplification method according to <8>, comprising the following steps.
(1) Step (2) in which a primer (A) is hybridized to a target nucleic acid sequence and a nucleic acid synthesis reaction starting from the 3 ′ end of the primer (A) is performed (2) A primer (B ) To the target nucleic acid sequence, the nucleic acid synthesis reaction starting from the 3 ′ end of the primer (B) (3) the primer (A) is hybridized to the nucleic acid synthesized in step (2), and the primer Step (4) of performing nucleic acid synthesis reaction starting from the 3 ′ end of (A) Primer (B) is hybridized to the target nucleic acid sequence to the nucleic acid synthesized in Step (3), and the 3 ′ end of primer (B) The nucleic acid synthesis reaction starting from step (5) The 3 ′ end of the nucleic acid synthesized in step (4) is hybridized to a region consisting of a complementary base sequence on the same strand, and the 3 ′ end is the starting point. Toss Nucleic acid synthesis reaction step (6) The nucleic acid synthesized in step (5) is hybridized to a region consisting of a complementary base sequence on the same strand, and the nucleic acid starting from the 3 'end <10> The method according to <8> or <9>, wherein a mutation recognition protein is further present in the <10> reaction system for performing the synthesis reaction.
<11> The method according to <10>, wherein the mutation recognition protein is MutS, MSH2 or MSH6, or a mixture of two or more thereof.
<12> The method according to any one of <8> to <11>, wherein a melting temperature adjusting agent is further present in the reaction system.
<13> The method according to <12>, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide, or glycerol, or a mixture of two or more thereof.
<14> The method according to any one of <8> to <13>, wherein the nucleic acid amplification reaction is performed isothermally.
<15> A method for detecting the presence or absence of a mutation in a target nucleic acid sequence, comprising the following steps.
(1) A step of performing an amplification reaction of a target nucleic acid sequence in a nucleic acid sample in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set according to any one of <1> to <7> are present ( 2) A method for detecting the presence or absence of methylation in a target nucleic acid sequence, comprising the following steps: <16> determining the presence or absence of a mutation in a target nucleic acid sequence based on the presence or absence of a product of a nucleic acid amplification reaction
(1) A step of replacing a methylated base in a nucleic acid sample containing a target nucleic acid sequence with another base (2) containing a primer containing a test methylation site <1> to <7> (3) A step of determining the presence or absence of methylation in the target nucleic acid sequence based on the presence or absence of the product of the nucleic acid amplification reaction <17 > The method according to <16>, wherein the treatment of converting the methylated base into another base in the step (2) is treatment with bisulfite.
<18> The method according to any one of <15> to <17>, wherein the method according to any one of <8 to <14> is used.
<19> A kit for nucleic acid amplification, comprising at least the primer set according to any one of <1> to <7>, a nucleic acid synthase, a substrate, and a buffer.
<20> Nucleic acid amplification according to <19> or <20>, comprising a nucleic acid amplification kit <21> melting temperature adjusting agent according to <19>, comprising a mutation recognition protein For kit.

  As shown in FIG. 2, the primer in the present invention is only required to have a single region that hybridizes to the template nucleic acid, and has the advantage of a high degree of freedom in primer design.

  The action mechanism of the nucleic acid amplification reaction according to the present invention will be described with reference to FIGS. 3 and 4 (steps 1 to 5) and FIG. 5 (steps 6 to 9).

Step 1: A nucleic acid sample containing a target nucleic acid sequence and a reagent are mixed and incubated at 60 ° C. The target nucleic acid is in a dynamic equilibrium at around 60 ° C., and either the first primer (FTP in FIG. 3) or the second primer (BTP in FIG. 4) hybridizes to the complementary portion of the target nucleic acid. By extending from there, the chain on one side is peeled off to be in a single-stranded state.

Step 2: Nucleic acid strand complementary to the template nucleic acid starting from the 3 ′ end of the F1 (B1 in FIG. 4) region of FIG. 3 of the FTP of FIG. 3 (BTP in FIG. 4) by the action of the strand displacement type nucleic acid synthase Is synthesized. Since this nucleic acid chain has self-complementary regions FT and FTc on the 5 ′ end side, it hybridizes within the self molecule to form a folded region.

Step 3: The F2 (B2 in FIG. 4) primer in FIG. 3 hybridizes to the outside of the FTP in FIG. 3 (BTP in FIG. 4). Nucleic acid synthesis is extended while peeling the nucleic acid chain from the previously synthesized FTP of FIG. 3 (BTP of FIG. 4).

Step 4: The nucleic acid strand synthesized from the F2 primer in FIG. 3 (B2 in FIG. 4) and the template nucleic acid become double stranded. The BTP (FIG. 4 FTP) in FIG. 3 hybridizes to the nucleic acid chain synthesized from the FTP of FIG. 3 (BTP in FIG. 4) peeled off in step 3 to become a single strand, and the BTP (FIG. 3) Complementary nucleic acid synthesis is performed starting from the 3 ′ end of FTP) in FIG.

Step 5: The B2 (F2 in FIG. 4) primer in FIG. 3 hybridizes outside the BTP in FIG. 3 (FTP in FIG. 4), and its 3 ′ end serves as a starting point by the action of the strand displacement type nucleic acid synthase. Nucleic acid synthesis is extended while stripping the nucleic acid strand from the BTP of FIG. 3 (FTP of FIG. 4) synthesized earlier. This process produces a double-stranded nucleic acid. In addition, since the nucleic acid chain synthesized from the BTP of FIG. 3 peeled off in the process of step 5 (FTP of FIG. 4) has a self-complementary sequence at both ends, it hybridizes within the self-molecule and forms a folded region. Thus, a dumbbell structure (5) is obtained. This structure becomes the starting point structure of the amplification cycle after step 6.

Step 6: The steps after the dumbbell structure in FIG. 3 will be described with reference to FIG. 5, but the same applies to the steps after the dumbbell structure in FIG. In the dumbbell-shaped structure (5) formed in step 5 (FIG. 3), nucleic acid synthesis using the FT region at the 3 ′ end as a starting point is elongated, and at this time, the loop on the 5 ′ end side is peeled off. Extended. Furthermore, since the F1c region of the loop on the 3 ′ end side is a single strand, FTP can hybridize, and the nucleic acid chain synthesized earlier from the FT region starts from the 3 ′ end of the F1 region. Nucleic acid synthesis extends while peeling.

Step 7: In Step 6, the nucleic acid chain extended from the FT region that has been peeled off by the nucleic acid strand extended and synthesized from FTP to become a single strand has a self-complementary region on its 3 ′ end side, so that a loop is formed. Form. Nucleic acid synthesis is started from the 3 ′ end of the BT region of this loop using the single-stranded self as a template. Then, this nucleic acid chain is elongated while peeling off the nucleic acid chain from the FTP in which the double-stranded part is formed, and the structure (7) is obtained.

Step 8: The nucleic acid strand synthesized from FTP in the process of Step 7 becomes a single strand and has self-complementary regions FT, FTc and BTc, BT at both ends thereof, so that it hybridizes within the self molecule. To form a folded region. This structure (8) is complementary to the structure (5) formed in step 5. In this structure (8), as in the case of structure (5), nucleic acid synthesis is performed using the 3 ′ end of the BT region as a starting point, and BTP is hybridized to the single-stranded B1c region. Nucleic acid synthesis is carried out while peeling off the nucleic acid strand from the BT region. As a result, the structure (5) is formed again through the same processes as those in the steps 5, 6, and 8.

Step 9: In the structure (7), BTP is hybridized to the B1 region which is a single strand, and a nucleic acid strand is synthesized while peeling off the double strand portion. As a result of these processes, amplification products having a structure that repeats complementary sequences on the same strand are synthesized with various chain lengths. By repeating the above reaction, a large amount of nucleic acid complementary to the target nucleic acid sequence in the template nucleic acid can be synthesized.

  The primer set according to the present invention comprises at least two kinds of primers that enable amplification of a target nucleic acid sequence by the nucleic acid amplification reaction as described above. That is, the primer (A) contained in the primer set of the present invention is complementary to the sequence region (b) (FTc and BTc in FIG. 2) on the same strand from the 5 ′ end to the 3 ′ end. Sequence region (a) (FT and BT in FIG. 2), sequence region complementary to region (a) (b), sequence region (c) on the 3 ′ end side that can hybridize to the target nucleic acid and serve as an amplification origin (FIG. 2 primers with F1 and B1).

  The primer used in the present invention is composed of deoxynucleotides and / or ribonucleotides, and has a chain length that allows base pairing with a target nucleic acid while maintaining the required specificity under given conditions. It is what has. In the case of the primer (A), the primer length used in the present invention includes self-complementary regions (a) and (b) in addition to the target nucleic acid sequence. Is preferred. Preferably it is 15-100 bases, More preferably, it is 25-50 bases. In the case of the primer (B), it is preferably 10 to 50 bases, more preferably 15 to 30 bases, in the same manner as the primer usually used in the PCR method.

  Each chain length of the regions (a) and (b) constituting the primer of the present invention is preferably 50 bases or less, more preferably 10 bases or less, and even more preferably 5 bases. If necessary, an intervening sequence that does not affect the reaction may be inserted between the region (a) and the region (b). The chain length of the intervening sequence is preferably 10 bases or less, more preferably 5 bases or less. It preferably has a sequence that is not complementary to the target nucleic acid sequence. As described above, the region (a) and the region (b) are preferably composed of complementary base sequences. For example, the regions (a) and (b) may be composed of only adenine (A) and thymine (T).

  Further, the region (c) constituting the primer used in the present invention may be any as long as it can serve as a starting point for template complementary strand synthesis in a nucleic acid amplification reaction. The chain length of the region (c) is preferably 10 to 50 bases, more preferably 15 to 30 bases.

  In the primer used in the present invention, it is preferable to set the Tm value of region (c) higher than the Tm value of region (a). The difference in Tm value between the region (c) and the region (a) is preferably 0 to 20 ° C, more preferably 5 to 10 ° C.

  Primers used in the present invention include oligonucleotide primers composed of unmodified deoxynucleotides and / or modified deoxynucleotides, oligonucleotide primers composed of unmodified ribonucleotides and / or modified ribonucleotides, unmodified deoxynucleotides and Chimeric oligonucleotide primers containing modified deoxynucleotides and unmodified ribonucleotides and / or modified ribonucleotides are also included.

  The primer set according to the present invention includes at least two types of primers (B) having a sequence region (d) complementary to the target nucleic acid sequence region (d ′) in addition to at least two types of the primers (A). (However, in the template nucleic acid sequence, the sequence region (d ′) is present on the 3 ′ end side of the sequence region (c ′)).

  The primers included in the primer set according to the present invention can be synthesized by any method that can be used for the synthesis of oligonucleotides, such as the phosphate triester method, the H-phosphonate method, the thiophosphonate method, and the like. The first and second primers can be easily obtained by, for example, synthesizing by the phosphoramidite method using a DNA synthesizer 394 type manufactured by ABI (Applied Biosystem Inc.).

  The primer used in the present invention can be labeled with a known labeling substance. Examples of labeling substances include binding ligands such as digoxin and biotin, enzymes, fluorescent substances, luminescent substances, and radioisotopes.

  In the present specification, when the expression “5 ′ end side” or “3 ′ end side” is simply used, it means the direction in the chain that is the template. Further, when the 3 ′ end side is the starting point for complementary strand synthesis, it means that the —OH group on the 3 ′ end side is the starting point for complementary strand synthesis.

  The “oligonucleotide” in the present invention indicates a polynucleotide having a particularly small number of bases. Generally, an oligonucleotide refers to a polynucleotide having a base number of 2 to 100, more generally about 2 to 50, and is called an oligonucleotide, but is not limited to these numerical values.

  In the present invention, the “target nucleic acid” or “target nucleic acid sequence” refers to a base sequence constituting a nucleic acid intended for synthesis in the present invention. In addition, when nucleic acid is amplified based on the nucleic acid amplification method of the present invention, the base sequence constituting the nucleic acid to be amplified is the target nucleic acid sequence.

  In general, the base sequence of a nucleic acid describes the base sequence of the sense strand from the 5 ′ end to the 3 ′ end. In addition to the base sequence of the sense strand, the target base sequence in the present invention includes its complementary strand, that is, the base sequence of the antisense strand. That is, the “target nucleic acid” or “target nucleic acid sequence” is used as a term meaning at least one of a base sequence to be amplified and its complementary strand.

  In the present invention, the target nucleic acid sequence is not limited to the base sequence of the nucleic acid used as a template. Therefore, the target nucleic acid sequence may be composed of the same base sequence as the template or may be a different base sequence. Mutations may be introduced into the base sequence of the template, or a target base sequence consisting of a base sequence obtained by joining a part of the base sequence of the template may be synthesized.

  In the present invention, the “template” means a nucleic acid on the side serving as a template for complementary strand synthesis. A complementary strand having a base sequence complementary to the template has a meaning as a strand corresponding to the template, but the relationship between them is merely relative. That is, the strand synthesized as a complementary strand can function as a template again.

  In the present invention, there are a case where the base sequence contained in the template nucleic acid is synthesized as it is as a target nucleic acid sequence, and a case where the purpose is to synthesize a nucleic acid having a base sequence different from the template nucleic acid. A nucleic acid having a base sequence different from that of the template nucleic acid is, for example, a case where a mutation is introduced into the base sequence contained in the template nucleic acid, or a region that is separated from the template nucleic acid is synthesized as a continuous base sequence. Can be mentioned. Furthermore, the target nucleic acid sequence in the present invention may be a base sequence in which base sequences derived from different nucleic acids are linked.

  In the present invention, “nucleic acid synthesis” means extension of a nucleic acid from an oligonucleotide that is the origin of synthesis. In addition to synthesis, when the production of other nucleic acids and the elongation reaction of the produced nucleic acids occur continuously, the series of reactions are collectively referred to as “nucleic acid amplification”.

  In the present invention, “hybridize” indicates that a nucleic acid forms a double helical structure by base pairing based on the Watson-Crick model. Therefore, even if the nucleic acid chain constituting the base pair bond is a single strand, it is hybridized if a complementary base sequence in the molecule forms a base pair bond. In the present invention, “annealing” and “hybridization” are synonymous in that a nucleic acid forms a double helix structure by base pairing.

  The term “complementary” used for characterization of the base sequence constituting the primer used in the present invention includes base sequences that are not completely complementary. That is, complementary to a certain sequence means a sequence that can hybridize under stringent conditions and can provide a starting point for complementary strand synthesis.

  The stringent conditions can be determined depending on the Tm value of the duplex of the primer according to the present invention and its complementary strand, the salt concentration of the hybridization solution, and the like. Sambrook, E .; F. Frisch, T .; Reference can be made to Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989).

  For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer to be used, the primer can be specifically hybridized to the target nucleic acid. Such a primer can be designed using commercially available primer construction software such as Primer 3 (manufactured by Whitehead Institute for Biomedical Research). According to a preferred embodiment of the present invention, a primer that hybridizes to a target nucleic acid comprises a sequence of all or part of a nucleic acid molecule complementary to the target nucleic acid.

  In the present invention, “nucleic acid” refers to DNA, RNA, or a chimeric molecule thereof. “Nucleic acid” is synonymous with the term “polynucleotide”. The nucleic acid may be natural or artificially synthesized. Moreover, even if it is a nucleotide derivative which consists of an artificial structure partially or entirely, it is contained in the nucleic acid of this invention as long as it can form a base pair bond. Moreover, the number of bases constituting the nucleic acid in the present invention is not particularly limited.

  The nucleic acid sample or template nucleic acid containing the target nucleic acid sequence used in the nucleic acid amplification reaction of the present invention is separated from, for example, blood, tissue, cells, biological samples such as animals and plants, or food, soil, drainage, etc. Isolated from the microorganism-derived sample. Isolation of the nucleic acid sample can be performed by any method, and examples thereof include a method using dissolution treatment with a surfactant, sonication, shaking and stirring using glass beads, and a French press. In addition, when an endogenous nuclease is present, it is preferable to purify the isolated nucleic acid. The purification of the nucleic acid can be performed by, for example, phenol extraction, chromatography, ion exchange, gel electrophoresis, density-dependent centrifugation, and the like.

  More specifically, the nucleic acid sample or template nucleic acid includes genomic DNA isolated by the above method, double-stranded nucleic acid such as a PCR fragment, cDNA prepared by reverse transcription from total RNA or mRNA, and the like. Any single stranded nucleic acid can be used. In the case of the above double-stranded nucleic acid, it can be used more optimally by carrying out denaturing to make it a single strand.

  The enzyme used in the reverse transcription reaction is not particularly limited as long as it has cDNA synthesis activity using RNA as a template. For example, avian myeloblastosis virus-derived reverse transcriptase (AMV RTase), Rous-related virus 2 Examples include reverse transcriptases of various origins such as reverse transcriptase (RAV-2 RTase) and Moloney murine leukemia virus-derived reverse transcriptase (MMLV RTase). In addition, a DNA polymerase having reverse transcription activity can also be used.

  In the nucleic acid amplification reaction, even when the template nucleic acid is a double-stranded nucleic acid, it can be used as it is in the reaction. However, if necessary, the primer nucleic acid can be denatured into a single strand to obtain a primer for the template nucleic acid. Annealing can also be performed efficiently. Increasing the temperature to about 95 ° C is a preferred nucleic acid denaturation method. As another method, it is possible to denature by raising the pH, but in this case, it is necessary to lower the pH in order to hybridize the primer to the target nucleic acid.

  The DNA polymerase used in the nucleic acid amplification reaction of the present invention only needs to have strand displacement activity (strand displacement ability), and any of room temperature, intermediate temperature, and heat resistance can be suitably used. . Further, this DNA polymerase may be either a natural body or a mutant with artificial mutation. Furthermore, it is preferable that this DNA polymerase has substantially no 5 ′ → 3 ′ exonuclease activity.

  Examples of the DNA polymerase used in the nucleic acid amplification reaction of the present invention include Bacillus stearothermophilus (hereinafter referred to as “B.st”), Bacillus caldotenax (hereinafter referred to as “B.ca”), and the like. Examples include mutants lacking the 5 ′ → 3 ′ exonuclease activity of thermophilic Bacillus-derived DNA polymerase, Klenow fragment of E. coli-derived DNA polymerase I, and the like.

  The DNA polymerase used in the nucleic acid amplification reaction of the present invention includes Vent DNA polymerase, Vent (Exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA Examples include polymerase, Z-Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, and Terminator DNA polymerase.

  Furthermore, in the nucleic acid amplification reaction of the present invention, by using a DNA polymerase having reverse transcription activity, for example, BcaBEST DNA polymerase, Bca (exo-) DNA polymerase, etc., reverse transcription reaction from total RNA or mRNA and cDNA are performed. The template DNA polymerase reaction can be carried out with one kind of polymerase. Alternatively, a DNA polymerase and a reverse transcriptase such as MMLV reverse transcriptase may be used in combination.

  Other reagents used in the nucleic acid amplification reaction of the present invention include, for example, salts such as magnesium chloride, magnesium acetate, magnesium sulfate, substrates such as dNTP, Tris-HCl buffer, tricine buffer, sodium phosphate buffer, potassium phosphate A buffer solution such as a buffer can be used. Further, additives such as dimethyl sulfoxide and betaine (N, N, N-trimethylglycine), acidic substances described in International Publication No. 99/54455 pamphlet, and cation complexes may be used.

  A mutation recognition protein may be added to the nucleic acid amplification reaction system of the present invention. As used herein, “mutation” refers to a different base (a base pair in the case of a double-stranded nucleic acid) in a target nucleic acid when compared to a control nucleic acid. A “mutation recognition protein” is a protein that binds to a mismatch site when a mismatch is contained in a double-stranded nucleic acid.

  In the present invention, “mismatch” means that a pair of base pairs selected from adenine (A), guanine (G), cytosine (C), and thymine (T) (uracil (U) in the case of RNA) is normal. It means that it is not a correct base pair (a combination of A and T, or a combination of G and C). Mismatches include not only one mismatch but also a plurality of consecutive mismatches, mismatches caused by insertion and / or deletion of one or more bases, and combinations thereof.

  By adding a mutation recognition protein (also referred to as a mismatch binding protein or a mismatch recognition protein) to the reaction system, the mutation recognition protein binds to the mutation portion in the double-stranded nucleic acid, and the nucleic acid amplification reaction is inhibited. This has the advantage that non-specific nucleic acid amplification is suppressed when there is a mutation in the double-stranded nucleic acid.

  Preferred examples of the mutation recognition protein include MutS, MSH2, and MSH6. However, the origin of the mutation recognition protein is not limited as long as it can recognize the mutation in the double-stranded nucleic acid.

  The mutation recognition protein used in the present invention may be a partial peptide of these proteins as long as the mutation in the double-stranded nucleic acid can be recognized. Furthermore, the mutation recognition protein may be a fusion protein with another protein such as glutathione-S-transferase.

  In addition, the mutation recognition protein is an amino acid sequence obtained by substituting, deleting, adding, and / or inserting one or more amino acids in the amino acid sequence of a natural protein as long as it can recognize a mutation in a double-stranded nucleic acid. It may be a protein (mutant) consisting of Such a mutant may occur in nature, but can be artificially prepared by appropriately using a known method.

  The mutation recognition protein can be prepared as a natural protein or as a recombinant protein by appropriately combining known methods such as anion exchange column, cation exchange column, gel filtration column chromatography, and ammonium sulfate fractionation. is there. When the recombinant protein has a high expression level, it can be easily prepared only by chromatography using a cation exchange column and a gel filtration column.

  The contact between the double-stranded nucleic acid and the mutation-recognizing protein in the method of the present invention is performed under conditions (for example, appropriate pH, solvent, ionic environment, temperature) that allow the protein to bind to the mutated region in the double-stranded nucleic acid. Done. Detailed conditions such as reaction temperature, salt concentration, ion type, buffer pH and the like can be appropriately adjusted.

  It is known that a mutation recognition protein may also bind to a single strand, and the binding of such a mutation recognition protein to a single-stranded nucleic acid is inhibited by the single-stranded binding protein. Therefore, when a mutation recognition protein is used in the nucleic acid amplification method of the present invention, it is preferable to use a single-stranded binding protein (SSB) in combination. The single chain binding protein can be any SSB in the art.

  In addition, mutation recognition proteins may bind to double-stranded nucleic acids that do not contain mismatches, and such erroneous binding can be prevented by activating the mutation recognition protein with an activator in advance. It has been known. Therefore, when a mutation recognition protein is used in the nucleic acid amplification method of the present invention, it is preferable to use a protein activated in advance by an activator.

  An activator for activating the mutation recognition protein can be appropriately selected by those skilled in the art and is not particularly limited, but is preferably ATP (adenosine 5'-triphosphate), ADP (adenosine 5'-). Diphosphate), ATP-γ-S (adenosine 5′-O- (3-thiotriphosphate)), AMP-PNP (adenosine 5 ′ [β, γ-imido] triphosphate) and the like. Or one of the nucleotides that can bind to the mutation recognition protein. Activation of the mutation recognition protein can be performed by incubating the mutation recognition protein and the active agent at room temperature for several seconds to several minutes.

  In the nucleic acid amplification reaction of the present invention, a melting temperature adjusting agent can be added to the reaction solution in order to increase the nucleic acid amplification efficiency. The melting temperature of a nucleic acid is generally determined by the specific nucleotide sequence of the duplex forming portion in the nucleic acid. By adding a melting temperature adjusting agent to the reaction solution, this melting temperature can be changed. Therefore, under a certain temperature, the intensity of double strand formation in the nucleic acid can be adjusted. A general melting temperature adjusting agent has an effect of lowering the melting temperature.

  By adding the melting temperature adjusting agent, it is possible to lower the melting temperature of the double-stranded forming part between two nucleic acids, in other words, to reduce the strength of the double-stranded formation. . Therefore, when such a melting temperature adjusting agent is added to the reaction solution in the nucleic acid amplification reaction, it is efficiently used in a GC-rich nucleic acid region that forms a strong double strand or a region that forms a complex secondary structure. The double-stranded portion can be made into a single strand, and this makes it easy for the next primer to hybridize to the target region after the extension reaction with the primer is completed, so that the nucleic acid amplification efficiency can be increased.

  The melting temperature adjusting agent used in the present invention and its concentration in the reaction solution are appropriately selected by those skilled in the art in consideration of other reaction conditions that affect the hybridization conditions, such as salt concentration, reaction temperature, etc. The Accordingly, the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof.

  In the nucleic acid amplification reaction of the present invention, an enzyme stabilizer can be added to the reaction solution. Thereby, since the enzyme in the reaction solution is stabilized, the amplification efficiency of the nucleic acid can be increased. The enzyme stabilizer used in the present invention may be any known in the art, such as glycerol, bovine serum albumin, saccharides, etc., and is not particularly limited.

  In the nucleic acid amplification reaction of the present invention, a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can also be added to the reaction solution. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid synthesis efficiency and amplification efficiency can be increased. Such a reagent may be any known in the art, and is not particularly limited, and is trehalose, sorbitol or mannitol, or a mixture of two or more thereof.

  The present invention provides a nucleic acid amplification method for performing an amplification reaction of a target nucleic acid sequence in a nucleic acid sample in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set of the present invention are present. The nucleic acid amplification reaction using the primer set according to the present invention can be performed isothermally. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. The substantially constant temperature condition means that not only the set temperature is accurately maintained, but also a temperature change that does not impair the substantial functions of the enzyme and the primer is allowed. For example, a temperature change of about 0 to 10 ° C. is acceptable.

  The nucleic acid amplification reaction under a certain temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained. In the nucleic acid amplification reaction, in order for the primer to anneal to the target nucleic acid, for example, the reaction temperature is preferably set to a temperature close to or lower than the Tm value of the primer. It is preferable to set the stringency level in consideration of the value. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C. In the nucleic acid amplification reaction, the amplification reaction is repeated until either the enzyme is inactivated or one of the reagents including the primer is used up.

  As described above, since the nucleic acid amplification reaction of the present invention can be carried out isothermally, it is less likely that a nucleic acid amplification enzyme (DNA polymerase or the like) is inactivated compared to the conventional PCR method. Therefore, the nucleic acid amplification reaction using the primer set according to the present invention is also effective for amplifying a target nucleic acid containing a non-natural nucleotide in which a nucleic acid amplification enzyme having no heat resistance is used. Here, `` non-natural nucleotide '' means a nucleotide containing a base other than the base (adenine, guanine cytosine, and thymine or uracil) contained in the natural nucleotide, which can be incorporated into the nucleic acid sequence empty, Examples thereof include xanthosines, diaminopyrimidines, isoG, isoC and the like.

  The enzyme used for amplification of a nucleic acid containing a non-natural nucleotide is not particularly limited as long as it can amplify such a target nucleic acid. Furthermore, a substance such as trehalose that improves the heat resistance of the nucleic acid amplification enzyme can also be added to the reaction solution, whereby the target nucleic acid sequence containing unnatural nucleotides can be efficiently amplified.

  In the nucleic acid amplification method of the present invention, a primer set according to the present invention is prepared for each target nucleic acid sequence, the plurality of primer sets are immobilized on a solid phase carrier in a form that can be distinguished from each other, and these immobilized primer sets The nucleic acid amplification reaction may be performed using This makes it possible to simultaneously amplify a plurality of target nucleic acids and detect the amplification products for each of them in a distinguishable form. Detection of the amplification product can be performed using an intercalator or the like. For example, by immobilizing a plurality of primers at specific positions on a planar solid phase carrier, the amplified target is detected depending on the position where the amplified product is detected after the nucleic acid amplification reaction and the detection of the amplified product. Nucleic acids can be identified.

  The presence of the amplification product obtained by the nucleic acid amplification method of the present invention can be detected by any other method. When performing other detection methods, when a primer set immobilized on a solid phase carrier is used, the solid phase carrier may be separated from the amplification product as necessary. Separation of the solid phase carrier from the amplification product can be performed by a method known in the art.

  Other detection methods include detection of amplification products by general agarose gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green. As yet another detection method, detection can be performed by using a labeled probe having a label such as biotin and hybridizing it to the amplification product. Biotin can be detected by binding to fluorescently labeled avidin, avidin bound to an enzyme such as peroxidase, and the like.

  Since nucleic acids synthesized by the nucleic acid amplification reaction of the present invention are composed of complementary base sequences, most of them form base pair bonds. This feature can be used to detect the product of the nucleic acid amplification reaction. If a fluorescent dye that is a double-strand specific intercalator such as ethidium bromide or cyber green is added to the reaction system in advance and the nucleic acid amplification reaction according to the present invention is performed, the fluorescence intensity increases as the number of products increases. Is observed. By monitoring this, the nucleic acid amplification reaction can be traced in real time.

  Since the amplified fragment obtained by the nucleic acid amplification reaction according to the present invention is composed of ordinary bases, it can be subcloned into an appropriate vector using a restriction enzyme site inside the amplified fragment. Furthermore, the amplified fragment can be treated with a restriction enzyme like RFLP, and can be widely used in the field of genetic testing. Further, the amplified fragment can be generated as containing an RNA polymerase promoter sequence, which makes it possible to synthesize RNA directly from the amplified fragment. The RNA synthesized in this way can be used as an RNA probe, siRNA or the like.

  In addition, in the nucleic acid amplification method according to the present invention, a base labeled with biotin or a fluorescent substance can be used as a substrate instead of the usual dNTP, whereby a DNA probe labeled with biotin or a fluorescent substance can be used. It is also possible to prepare. Furthermore, it is also possible to confirm the presence or absence of an amplification product through some structure such as biotin or a fluorescent substance.

  Using a nucleic acid amplification reaction using the primer set according to the present invention, it is possible to determine the presence or absence of a mutation in a target nucleic acid sequence in a nucleic acid sample. For this purpose, the primer set of the present invention is designed so that the test mutation site is contained in a primer (preferably in the region (c) constituting the primer, more preferably in the vicinity of the 3 ′ end of the region (c)). Thus, by confirming the presence or absence of the amplification product, it is possible to determine the presence or absence of the mutation.

Accordingly, the present invention provides a method for detecting the presence or absence of a mutation in a target nucleic acid sequence, comprising the following steps.
(1) A step of performing an amplification reaction of the target nucleic acid sequence in the nucleic acid sample in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set of the present invention are present (2) Depending on the presence or absence of a product of the nucleic acid amplification reaction, A step of determining the presence or absence of mutation in the target nucleic acid sequence

  In the mutation detection method according to the present invention, when a primer set that causes a mismatch with the template nucleic acid due to the presence of the target mutation is used, the presence of the amplification product after the nucleic acid amplification reaction indicates the absence of the mutation. Absence or reduction indicates the presence of the mutation. On the other hand, if an amplification product is obtained using a primer set that mismatches with the template nucleic acid due to the absence of the target mutation, it can be determined that the mutation is present in the nucleic acid sample, and conversely the amplification product Can not be obtained, it can be determined that the mutation does not exist. Here, “decrease in amplification product” indicates that the amount of amplification product obtained is reduced compared to the amount of amplification product obtained when the target nucleic acid sequence is present in the nucleic acid sample.

  Further, it is possible to determine the presence or absence of methylation in a target nucleic acid sequence in a nucleic acid sample using a nucleic acid amplification reaction using the primer set according to the present invention. For this purpose, the methylated base in the nucleic acid sample is replaced with another base before the nucleic acid amplification reaction, and then the test methylation site is a primer (preferably a region constituting the primer). The nucleic acid amplification reaction is performed using the primer set of the present invention designed to be included in (c), more preferably in the vicinity of the 3 ′ end of the region (c). The presence or absence of mutation can be determined by confirming the presence or absence of the amplification product.

  The treatment for substituting a methylated base with another base is not particularly limited, and any method known in the art may be used as appropriate. When the target sample nucleic acid is treated with bisulfite, unmethylated C (cytosine) is converted to U (uracil), whereas methylated C is not converted. Therefore, after the bisulfite treatment, an amplification reaction is performed using a primer set corresponding to each base, whereby a methylated sample and an unmethylated sample can be separated.

Accordingly, the present invention provides a method for detecting the presence or absence of methylation in a target nucleic acid sequence, comprising the following steps.
(1) A step of replacing a methylated base in a nucleic acid sample containing a target nucleic acid sequence with another base (2) Using the primer set of the present invention containing a primer containing a test methylation site And (3) a step of determining the presence or absence of methylation in the target nucleic acid sequence based on the presence or absence of a product of the nucleic acid amplification reaction.

  In the methylation detection method according to the present invention, when a primer set that mismatches with the template nucleic acid due to the presence of mutation at the test methylation site is used, the presence of the amplification product after the nucleic acid amplification reaction indicates the absence of methylation. The absence or reduction of amplification product indicates the presence of the methylation. On the other hand, when an amplification product is obtained using a primer set that mismatches with the template nucleic acid due to the absence of mutation at the test methylation site, it can be determined that the methylation is present in the nucleic acid sample, Conversely, when no amplification product is obtained, it can be determined that the methylation does not exist.

  The mutation detection method and methylation detection method of the present invention can improve their specificity by using a mutation recognition protein. Specifically, when the test nucleic acid contained in the nucleic acid sample has a nucleotide different from the target nucleic acid sequence at the test mutation site or test methylation site, it hybridizes to the target nucleic acid in the primer of the present invention and amplifies it. Since the hybridization to the test nucleic acid in the region (c) that can serve as a starting point is hindered, an amplification product cannot be obtained or the amount of the amplification product is reduced.

  However, the hybridization may not be completely prevented, in which case a small amount of heteroduplex structure results in these sequences. The term “heteroduplex structure” as used herein refers to a duplex structure that is substantially complementary and includes a non-complementary region by having one or more mismatches. . Such a heteroduplex structure generates an erroneous amplification product. In this case, by adding the mutation recognition protein to the reaction solution used for the nucleic acid amplification reaction, the mutation recognition protein binds to the heteroduplex structure, and the subsequent amplification reaction is hindered. Therefore, by using the mutation recognition protein, it becomes possible to prevent generation of erroneous amplification products.

  In order to carry out the nucleic acid amplification method or nucleic acid detection method according to the present invention, necessary reagents can be put together into a kit. Accordingly, the kit according to the present invention comprises the primer set according to the present invention.

  Furthermore, when at least one of the primer sets according to the present invention includes a site capable of binding to a solid phase carrier, the kit according to the present invention preferably further comprises the solid phase carrier. In addition, when the substrate used in the nucleic acid amplification reaction includes a site capable of binding to the solid phase carrier, the kit according to the present invention preferably further includes the solid phase carrier. The kit according to the present invention may further contain the above-mentioned reagents such as a nucleic acid synthesizing enzyme such as DNA polymerase, a substrate such as dNTP, a buffer solution, a mutation recognition protein, a melting temperature adjusting agent, a reaction container, and instructions. By using such a kit, a nucleic acid amplification reaction can be carried out simply by adding a template nucleic acid or a nucleic acid sample to the reaction vessel and keeping the reaction vessel at a constant temperature.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to these examples.

  A target nucleic acid sequence in the human β-actin gene was amplified using a primer set including a primer having two self-complementary sequences.

(1) Preparation of nucleic acid sample solution containing target nucleic acid sequence 100 ng of HumanGenomic cDNA (manufactured by Clontech) was heated at 98 ° C. for 3 minutes to make a single strand, and then the amplification of the sequence in the β-actin gene was performed under the following conditions: I went there. As a negative control, a sample in which water was heated under the same conditions as described above was also prepared.

<Primer>
The primer was designed using a human β-actin gene (GenBank accession number: AC006483, target nucleic acid sequence: nucleotides 171170 to 171282 from the 5 ′ end) as a template. Both forward primer <1> (SEQ ID NO: 1) and reverse primer <2> (SEQ ID NO: 2) are complementary to the template and are designed to have a folded structure. Consists of. Moreover, outer primer <3> and <4> (OF, OR) (sequence number 3, 4) were designed on the outer side of each of forward primer <1> and reverse primer <2>.

The primer sequences are shown below. The folded structure in the forward primer and reverse primer is underlined.
Forward primer <1>:
5'- tttatatatatatataaa GGGCATGGGTCAGAAGGATT-3 '(SEQ ID NO: 1)
Reverse primer <2>:
5'- tttatatatatatataaa CATGTCGTCCCAGTTGGTGA-3 '(SEQ ID NO: 2)
Outer primer <3> (OF)
5'-GGGCTTCTTGTCCTTTCCTTC-3 '(SEQ ID NO: 3)
Outer primer <4> (OR)
5′-CCACACGCAGCTCATTGTAG-3 ′ (SEQ ID NO: 4)

(2) Nucleic acid amplification reaction The amplification reaction was carried out by reacting at 60 ° C for 90 minutes with the composition of the reaction solution shown below.

<Composition of reaction solution>
10 × Bst Buffer (DF) 2.5 μl
100 mM MgSO ₄ 1.5 μl
10% (v / v) Tween 20 0.25 μl
100% DMSO 1.25 μl
25 mM dNTP 1.4 μl each
SYBR Green I 0.5 μl
50 μM primer <1> 1.6 μl
50 μM primer <2> 1.6 μl
50 μM primer <3> 0.2 μl
50 μM primer <4> 0.2 μl
Bst. Polymerase 1.0 μl
TaqMutS 1.0μl
Nucleic acid fragment sample solution obtained in (1) (100 ng / μl) 1.0 μl
Purified water 11.0μl

Total volume 25.0μl

(3) Detection of amplification product 5 μl of the reaction solution after the amplification reaction in (2) above was applied to a 2% agarose gel (1 × TAE) and subjected to electrophoresis to confirm the amplification product. The result is shown in FIG.

  Amplification products were observed only from samples derived from nucleic acids. In addition, a clear peak was observed in the vicinity of 120 bp, which was predicted from the primer design information, and it was found that the targeted amplification product was obtained.

The schematic of the primer used for LAMP method is shown. 1 shows a schematic diagram of primers used in the nucleic acid amplification method according to the present invention. The basic reaction principle of the nucleic acid amplification method according to the present invention will be described. The basic reaction principle of the nucleic acid amplification method according to the present invention will be described. The basic reaction principle of the nucleic acid amplification method according to the present invention will be described. The amplification product obtained by the amplification reaction was confirmed by electrophoresis on a 2% agarose gel (1 × TAE). The upper row shows a nucleic acid sample, the middle row shows water, and the lower row shows a 100 bp ladder. (Example 1)

Claims (21)

  From the 5 ′ end to the 3 ′ end, the nucleic acid sequence region (a), the nucleic acid sequence region (b) complementary to the sequence of the region (a), and the region complementary to the region (c ′) in the target nucleic acid sequence A primer set for amplifying a target nucleic acid sequence, comprising at least two kinds of primers (A) containing a nucleic acid sequence region (c).   The primer (B) having a nucleic acid sequence region (d) complementary to the region (d ') in the target nucleic acid sequence in addition to the primer (A) is included. Primer set for target nucleic acid sequence amplification (however, in the target nucleic acid sequence, the region (d ′) is present on the 3 ′ end side from the region (c ′)).   The primer set according to claim 1 or 2 for use in amplification of a target nucleic acid sequence under isothermal conditions.   The primer set according to any one of claims 1 to 3, wherein each of the regions (a) and (b) comprises 50 bases or less.   The sequence present between the regions (a) and (b) consists of 10 bases or less, or the regions (a) and (b) are adjacent to each other. The primer set according to any one of the above.   The primer set according to any one of claims 1 to 5, wherein the regions (a) and (b) are composed of only adenine (A) and thymine (T).   The primer set according to any one of claims 1 to 6, wherein the Tm value of the region (c) is higher than the Tm value of the region (a).   A nucleic acid amplification method for performing an amplification reaction of a target nucleic acid sequence in a nucleic acid sample in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set according to any one of claims 1 to 7 are present. The nucleic acid amplification method according to claim 8, comprising the following steps.
(1) Step (2) in which a primer (A) is hybridized to a target nucleic acid sequence and a nucleic acid synthesis reaction starting from the 3 ′ end of the primer (A) is performed (2) A primer (B ) To the target nucleic acid sequence, the nucleic acid synthesis reaction starting from the 3 ′ end of the primer (B) (3) the primer (A) is hybridized to the nucleic acid synthesized in step (2), and the primer Step (4) of performing nucleic acid synthesis reaction starting from the 3 ′ end of (A) Primer (B) is hybridized to the target nucleic acid sequence to the nucleic acid synthesized in Step (3), and the 3 ′ end of primer (B) The nucleic acid synthesis reaction starting from step (5) The 3 ′ end of the nucleic acid synthesized in step (4) is hybridized to a region consisting of a complementary base sequence on the same strand, and the 3 ′ end is the starting point. Toss Nucleic acid synthesis reaction step (6) The nucleic acid synthesized in step (5) is hybridized to a region consisting of a complementary base sequence on the same strand, and the nucleic acid starting from the 3 'end Process for performing synthesis reaction   The method according to claim 8 or 9, wherein a mutation recognition protein is further present in the reaction system.   The method according to claim 10, wherein the mutation recognition protein is MutS, MSH2 or MSH6, or a mixture of two or more thereof.   The method according to any one of claims 8 to 11, wherein a melting temperature adjusting agent is further present in the reaction system.   The method according to claim 12, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.   The method according to any one of claims 8 to 13, wherein the nucleic acid amplification reaction is performed isothermally. A method for detecting the presence or absence of a mutation in a target nucleic acid sequence, comprising the following steps.
(1) A step of performing an amplification reaction of a target nucleic acid sequence in a nucleic acid sample in a reaction system in which the nucleic acid sample containing the target nucleic acid sequence and the primer set according to any one of claims 1 to 7 are present (2) A step of determining the presence or absence of a mutation in a target nucleic acid sequence based on the presence or absence of a product of a nucleic acid amplification reaction A method for detecting the presence or absence of methylation in a target nucleic acid sequence, comprising the following steps.
(1) A step of replacing a methylated base in a nucleic acid sample containing a target nucleic acid sequence with another base (2) A primer containing a test methylation site (3) A step of determining the presence or absence of methylation in the target nucleic acid sequence based on the presence or absence of a product of the nucleic acid amplification reaction, using the primer set according to claim 1   The method according to claim 16, wherein the treatment for converting the methylated base into another base in the step (2) is treatment with bisulfite.   The method according to any one of claims 15 to 17, characterized in that the method according to any one of claims 8 to 14 is used.   A kit for nucleic acid amplification, comprising at least the primer set according to any one of claims 1 to 7, a nucleic acid synthase, a substrate, and a buffer solution.   The kit for nucleic acid amplification according to claim 19, comprising a mutation recognition protein.   The nucleic acid amplification kit according to claim 19 or 20, further comprising a melting temperature adjusting agent.