JP2007325534A - METHOD FOR ISOTHERMAL AMPLIFICATION OF NUCLEIC ACID UTILIZING RecA PROTEIN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for amplifying a nucleic acid by which the nucleic acid can be amplified with high sequence specificity. <P>SOLUTION: The method for amplifying a target nucleic acid sequence in a template nucleic acid by a nucleic acid amplifying reaction under isothermal condition comprises the following steps: (a) a step of preparing the template nucleic acid containing the target nucleic acid sequence, (b) a step of preparing an RecA protein, (c) a step of preparing a primer set for affording the amplification of the target nucleic acid sequence under isothermal condition and (d) a step of carrying out the nucleic acid amplification reaction under isothermal condition with the primer set in the presence of the template nucleic acid and the RecA protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Background of the Invention

The present invention relates to a method for amplifying a nucleic acid sequence useful in the field of genetic engineering, and more specifically, a method for isothermal amplification of a nucleic acid sequence using a strand displacement reaction, and a method for detecting a mutation using this method. It is about.

BACKGROUND ART In the field of genetic engineering, analysis based on complementarity of nucleic acid sequences is known as a method that can directly analyze genetic characteristics. In such an analysis, when the amount of the target gene present in the sample is small, it is generally not easy to detect the target gene, so it is necessary to amplify the target gene itself in advance.

  Amplification of the target gene (nucleic acid amplification) is mainly performed by an enzymatic method using DNA polymerase. Major examples of such enzymatic methods include the polymerase chain reaction method (PCR method; US Pat. No. 4,683,195, US Pat. No. 4,683,202 and US Pat. No. 4,800,159), and There is a reverse transcription PCR method (RT-PCR method; Trends in Biotechnology 10, pp146-152, 1992), which combines a PCR method and a reverse transcriptase reaction. These methods consist of three steps: dissociation (denaturation) of a double-stranded nucleic acid serving as a template into a single-stranded nucleic acid, annealing of a primer into a single-stranded nucleic acid, and synthesis of a complementary strand from the primer (extension). By repeating the reaction, the target gene can be amplified from DNA or RNA. In these methods, a total of three steps are required to adjust the reaction solution to a temperature suitable for each of the above three steps.

  In recent years, nucleic acid amplification methods that can be performed in an isothermal state have been developed. Examples of such a method include a strand displacement amplification (SDA) method, a self-sustained sequence replication (3SR) method, and Japanese Patent No. 2650159 described in Japanese Patent Publication No. 7-114718. Nucleic acid sequence amplification (NASBA) method, TMA (transcription-mediated amplification) method, Q beta replicase method described in Japanese Patent No. 2710159, US Pat. No. 5,824,517 Various modified SDA methods described in WO99 / 09211 or WO95 / 25180, LAMP method (Loop-Mediated Isothermal Amplification) described in WO00 / 28082, and international publication ICAN method (Isotherm) described in pamphlet No. 02/16639 al and Chimeric primer-initiated Amplification of Nucleic acids), the method described in International Publication No. 2004/040019 pamphlet, and the SMAP method described in International Publication No. 2005/063977 pamphlet. All of the reactions involved in these isothermal nucleic acid amplification methods proceed simultaneously in a reaction mixture maintained at a constant temperature.

  In the LAMP method described in WO 00/28082 pamphlet (Patent Document 1), four types of primers are required, and the target gene can be amplified by recognizing six regions. That is, in this method, first, the first primer anneals to the template strand to cause an extension reaction, and then the first primer undergoes a strand displacement reaction with a second primer designed upstream of the first primer. The extended strands from the primers are separated from the template strand. At this time, a stem-loop structure is formed at the 5 'end portion of the extended strand due to the structure of the first primer extension product that has been peeled off. A similar reaction is also performed on the other strand of the double-stranded nucleic acid or on the 3 ′ end side of the first primer extension product that has been peeled off. By repeating these reactions, the target nucleic acid Amplified. In this method, an amplification product that forms a stem-loop structure at the 3 ′ end is obtained by complementary strand synthesis to the primer extension strand, and complementary strand synthesis with the 3 ′ end as the synthesis origin occurs continuously. A long amplification product is obtained in which the nucleic acid sequences are linked via primer sequences.

  In the method described in the pamphlet of International Publication No. 2004/040019 (Patent Document 2), a pair of loop-forming primers capable of forming a stem-loop structure at the 5 'end portion of the primer extension strand is used. This loop-forming primer was annealed to the template and passed through a primer extension reaction, and then automatically looped back to the extension strand at the 5 ′ end of the extension strand to form a loop, thereby becoming a single-stranded state. Allows the same primer to anneal to a region on the template. Even in this method, complementary strand synthesis to the primer extension strand yields an amplification product that forms a stem-loop structure at the 3 'end, and complementary strand synthesis with the 3' end as the synthesis origin occurs continuously. A long amplification product is obtained in which the nucleic acid sequences are linked via primer sequences.

  In the SMAP method described in International Publication No. 2005/063977 (Patent Document 3), a loop-forming primer described in International Publication No. 2004/040019 and a mutually complementary sequence are linked on a single strand. A primer pair consisting of a folding primer containing a folding sequence at the 5 ′ end is used. In this method, an amplification product that forms a stem-loop structure or a folded structure at the 3 ′ end is obtained by complementary strand synthesis to the primer extension strand, and complementary strand synthesis with the 3 ′ end as the starting point of synthesis occurs continuously. A long amplification product is obtained in which many target nucleic acid sequences are linked via primer sequences.

  On the other hand, in nucleic acid amplification techniques using primers such as the PCR method, there is a problem that the sequence specificity cannot be sufficiently ensured, such as insufficient amplification of the target nucleic acid sequence and increase of amplification products other than the target. May occur.

  In order to improve the sequence specificity in the nucleic acid amplification reaction, it has been reported that a DNA-binding protein derived from E. coli that binds to single-stranded DNA is added to the reaction solution with respect to the PCR method. For the same purpose, RecA protein derived from Escherichia coli and the like and ATPγ-s are added to the PCR reaction solution (Patent Document 4: International Publication No. 91/17267 pamphlet), RecA protein derived from Thermus aquaticus and A method of adding ATP (Patent Document 4: WO 91/17267 pamphlet), a method of adding RecA protein derived from Thermus thermophilus (Patent Document 5: JP 2005-110621 A), and a method derived from Thermus thermophilus A method of adding RecA protein and ATPγ-s (Patent Document 6: International Publication No. 2004/027060 pamphlet) has been reported.

  In recent years, diagnostic techniques for rapidly detecting gene information such as gene insertions and deletions have become very important. Emphasis is placed on technologies that can be used for detection. Thus, there remains a need for nucleic acid amplification methods that have high sequence specificity and high amplification efficiency.

International Publication No. 00/28082 Pamphlet International Publication No. 2004/040019 Pamphlet International Publication No. 2005/063977 Pamphlet International Publication No. 91/17267 Pamphlet JP 2005-110621 A International Publication No. 2004/027060 Pamphlet

Summary of the Invention

  The present inventors have found that the sequence specificity of nucleic acid amplification is remarkably improved by carrying out an isothermal nucleic acid amplification reaction in the presence of RecA protein. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide a simple nucleic acid amplification method that enables nucleic acid amplification with high sequence specificity.

  The nucleic acid amplification method according to the present invention is a method of amplifying a target nucleic acid sequence in a template nucleic acid by an isothermal nucleic acid amplification reaction, comprising: (a) preparing a template nucleic acid containing the target nucleic acid sequence; ) A step of preparing RecA protein, (c) a step of preparing a primer set that enables amplification of the target nucleic acid sequence under isothermal conditions, and (d) the primer set in the presence of the template nucleic acid and RecA protein. Comprising a step of performing a nucleic acid amplification reaction under isothermal conditions.

  According to the present invention, it is possible to accurately amplify a target nucleic acid sequence contained in a template by a simple operation. Therefore, according to the present invention, it is possible to quickly, conveniently and accurately detect a target nucleic acid in a nucleic acid sample, detect a mutation in a gene, particularly a single nucleotide mutation, and the like.

Detailed description of the invention

  In the present invention, “RecA protein” is a kind of protein involved in homologous recombination of DNA, exhibits the action of ATPase, partially unwinds double-stranded DNA, and has a single-stranded complementary strand. A protein that induces recombination of DNA by forming hydrogen bonds with it. In the present invention, “RecA protein” includes mutants, modified proteins, and fragments having the same functions as those of natural RecA protein. As a RecA protein, it can select from various things well-known in this technical field, for example, RecA protein derived from colon_bacillus | E._coli, Thermus thermophilus, Thermus aquaticus etc. can be used. For such RecA protein, and mutants, modified proteins, and fragments thereof, see, for example, International Publication No. 91/17267 Pamphlet, Japanese Patent Application Laid-Open No. 2005-110621, International Publication No. 2004/027060 Pamphlet, etc. I want.

  The concentration of RecA protein in the reaction solution is appropriately determined by those skilled in the art depending on the type of the reaction solution, but is preferably 0.001 to 5000 ng / μL, more preferably 0.01 to 500 ng / μL, and still more preferably 0.8. 1 to 50 ng / μL.

  Furthermore, RecA protein is known to be activated in the presence of ATP (adenosine 5'-triphosphate) or its analogs. Therefore, according to a preferred embodiment of the present invention, the nucleic acid amplification reaction of the present invention is performed in the presence of ATP and / or an ATP analog. The ATP analog may be any structural analog of ATP that can activate the RecA protein in the same manner as ATP, and can be appropriately selected by those skilled in the art. Examples of such ATP analogs include ATPγ-s, rATP, and dATP, and preferably ATPγ-s. In the nucleic acid amplification reaction in the present invention, one kind of substance selected from ATP and ATP analogs may be used alone, or two or more kinds of substances may be used in combination.

  The total concentration of ATP and ATP analog in the reaction solution is appropriately determined by those skilled in the art so that the RecA protein is sufficiently activated, but is preferably 0.001 to 1000 mM, more preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM.

  The primer set used in the present invention enables amplification of a target nucleic acid sequence under isothermal conditions. Various nucleic acid amplification methods carried out under isothermal conditions are known in the art, such as the SDA method (Japanese Patent Publication No. 7-114718), the improved SDA method (US Pat. No. 5,824,517); International Publication No. 99/09211 pamphlet; International Publication No. 95/25180 pamphlet), NASBA method (Japanese Patent No. 2650159 publication), LAMP method (International Publication No. 00/28082 pamphlet), ICAN method (International Publication No. 02/16639 pamphlet), the method described in WO 2004/040019 pamphlet, the SMAP method described in WO 2005/063977 pamphlet, and the like. Therefore, those skilled in the art can design an appropriate primer set used in the present invention according to the type of nucleic acid amplification method to be used.

  According to a preferred embodiment of the present invention, at least one primer included in the primer set is a nucleic acid strand obtained by an extension reaction of the primer on a template, and the 5 ′ end portion of the nucleic acid strand is on the same strand. It is assumed to be capable of hybridizing (hereinafter referred to as “folding primer”). Here, the “nucleic acid chain” means any of a DNA chain, an RNA chain, and a DNA / RNA chain, preferably a DNA chain. The primer set used in the present invention may include a plurality of types of folding primers. For example, it is preferable to design one folding primer as a forward primer and the other folding primer as a reverse primer.

  In the present invention, the “target nucleic acid” or “target nucleic acid sequence” means not only the nucleic acid to be amplified or the sequence itself, but also a complementary sequence or a nucleic acid having the sequence.

  In the present invention, “hybridize” means that a part of the primer according to the present invention hybridizes to a target nucleic acid under stringent conditions and does not hybridize to a nucleic acid molecule other than the target nucleic acid. Stringent conditions can be determined depending on the melting temperature Tm (° C.) of the duplex between the primer according to the present invention and its complementary strand, the salt concentration of the hybridization solution, etc., for example, J. Sambrook, Reference can be made to EF Frisch, T. 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.

  Those skilled in the art can appropriately design the folded primer included in the primer set. The folded primer typically comprises a sequence that hybridizes to the template at its 3 ′ end portion, and a sequence that hybridizes to any region on the extended strand of the primer on the 5 ′ side. Can be. Such a folding primer can be appropriately designed in accordance with, for example, the descriptions in International Publication No. 00/28082, International Publication No. 2004/040019, International Publication No. 2005/063977, and the like.

  According to a further preferred embodiment of the present invention, the at least one primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion. And a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) that is present 5 ′ to the sequence (A) in the target nucleic acid sequence. 5 'side. This primer is an example of the folding primer, and is hereinafter referred to as “loop forming primer”.

  The action mechanism of nucleic acid synthesis using a loop-forming primer is schematically shown in FIG. First, a target nucleic acid sequence in a nucleic acid to be a template is determined, and a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence and a sequence (B) existing on the 5 ′ side of the sequence (A) are determined. . The loop-forming primer includes a sequence (Ac ′), and further includes a sequence (B ′) on the 5 ′ side thereof. The sequence (Ac ′) hybridizes to the sequence (A), and the sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B). Here, the loop-forming primer may contain an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence (B ′). When such a primer is annealed to the template nucleic acid, the sequence (Ac ′) in the primer is hybridized to the sequence (A) of the target nucleic acid sequence (FIG. 1 (a)). When the primer extension reaction occurs in this state, a nucleic acid containing a complementary sequence of the target nucleic acid sequence is synthesized. Then, the sequence (B ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Bc) present in the nucleic acid. A loop structure is formed. As a result, the sequence (A) on the template nucleic acid becomes a single strand, and another primer having the same sequence as the previous loop-forming primer hybridizes to this portion (FIG. 1 (b)). Thereafter, an extension reaction from the newly hybridized loop-forming primer occurs by the strand displacement reaction, and at the same time, the previously synthesized nucleic acid is separated from the template nucleic acid (FIG. 1 (c)).

  In the above mechanism of action, the phenomenon in which the sequence (B ′) hybridizes to the sequence (Bc) typically occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from its terminal or other relatively unstable part. In the double-stranded nucleic acid generated by the extension reaction using the loop-forming primer, the base pair at the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double-stranded nucleic acid is maintained as a whole. In such a state, when a sequence complementary to the dissociated portion of the terminal is present on the same strand, a stem loop structure can be formed as a metastable state. Although this stem-loop structure does not exist stably, the same other primer binds to the complementary strand part (sequence (A) on the template nucleic acid) exposed by the formation of the structure, and the polymerase immediately extends. By performing the above, a previously synthesized strand is replaced and released, and at the same time, a new double-stranded nucleic acid can be generated.

  The design criteria for the loop-forming primer in a further preferred embodiment of the present invention are as follows. First, in order for a new primer to efficiently anneal to the template nucleic acid after the complementary strand of the template nucleic acid is synthesized by extension of the primer, the template nucleic acid is formed by forming a stem loop structure at the 5 ′ end of the synthesized complementary strand. The part of the sequence (A) above needs to be a single strand. For this purpose, the difference (X−Y) between the base number X of the sequence (Ac ′) and the base number Y of the region sandwiched between the sequence (A) and the sequence (B) in the target nucleic acid sequence is expressed as X The ratio (X−Y) / X to is important. However, it is not necessary to make a single strand up to the portion which is present 5 ′ from the sequence (A) on the template nucleic acid and which is not related to primer hybridization. In addition, in order to efficiently anneal a new primer to the template nucleic acid, it is necessary to efficiently perform the above-described stem loop structure formation. For efficient stem loop structure formation, that is, efficient hybridization between the sequence (B ′) and the sequence (Bc), the distance between the sequence (B ′) and the sequence (Bc) ( X + Y) is important. In general, the optimum temperature for the primer extension reaction is at most around 72 ° C., and at such a low temperature, it is difficult for the extended strand to dissociate over a long region. Therefore, in order for the sequence (B ′) to efficiently hybridize to the sequence (Bc), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (B ′) to hybridize to the sequence (Bc) and to make the portion of the sequence (A) on the template nucleic acid a single strand, the sequence (B ′) and the sequence (Bc) It is considered that a larger number of bases between is preferred.

  From the above viewpoints, the loop-forming primer according to a further preferred embodiment of the present invention has a (X- Y) / X is −1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, preferably 0.75 or less, It is designed to be preferably 0.50 or less, more preferably 0.25 or less. Further, (X + Y) is preferably 15 or more, more preferably 20 or more, further preferably 30 or more, and is preferably 50 or less, more preferably 48 or less, and even more preferably 42 or less.

  When an intervening sequence (the number of bases is Y ′) exists between the sequence (Ac ′) and the sequence (B ′) constituting the primer, the loop-forming primer according to a further preferred embodiment of the present invention is {X- (YY ')} / X is -1.00 or more, preferably 0.00 or more, more preferably 0.05 or more, more preferably 0.10 or more, and 1.00 or less, It is designed to be preferably 0.75 or less, more preferably 0.50 or less, and still more preferably 0.25 or less. Further, (X + Y + Y ′) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 100 or less, more preferably 75 or less, and even more preferably 50 or less.

  The loop-forming primer has a chain length that allows base pairing with a target nucleic acid while maintaining the necessary specificity under given conditions. The primer has a chain length of preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. The lengths of the sequence (Ac ′) and the sequence (B ′) constituting the first primer are preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides, respectively. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Ac ′) and the sequence (B ′).

  According to another preferred embodiment of the present invention, at least one primer contained in the primer set according to the present invention has a sequence (Ac ′) that hybridizes to the sequence (A ′) of the 3 ′ terminal portion of the target nucleic acid sequence. It is assumed that it contains a folded sequence (B-Bc ') on the same strand containing two nucleic acid sequences that are hybridized to each other on the 5' side of the sequence (Ac '). The This primer is an example of the folding primer, and is hereinafter referred to as “folding primer”.

  The structure of such a folding primer is, for example, as shown in FIG. 2, but is not limited to the sequence and the number of nucleotides shown in FIG. The length of the sequence (Ac ′) constituting the folding primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (B-Bc ′) is preferably 2 to 1000 nucleotides, more preferably 2 to 100 nucleotides, still more preferably 4 to 60 nucleotides, and even more preferably 6 to 40 nucleotides. The number of nucleotides of base pairs formed by hybridization within the sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, still more preferably 2 to 30 bp, and further preferably 3 to 20 bp. The nucleotide sequence of the folded sequence (B-Bc ′) may be any sequence, and is not particularly limited, but is preferably a sequence that does not hybridize to the target nucleic acid sequence. If necessary, an intervening sequence that does not affect the reaction may be inserted between the sequence (Ac ′) and the folded sequence (B-Bc ′).

  The folding primer can act similarly to the mechanism of action described for the loop-forming primer. In general, when a double-stranded nucleic acid is dissociated into a single strand, partial dissociation starts from its terminal or other relatively unstable part. In the double-stranded nucleic acid generated by the extension reaction with the folding primer, the base pair of the terminal portion is in an equilibrium state of dissociation and binding at a relatively high temperature, and the double-stranded nucleic acid is maintained as a whole. At the terminal portion thus dissociated, a new folding primer anneals to the template, from which complementary strand synthesis proceeds with strand displacement.

  These loop forming primers and folding primers can be used in any combination. That is, two types of loop forming primers may be used as the forward primer and the reverse primer, two types of folding primers may be used as the forward primer and the reverse primer, or the loop forming primer and the folding primer are forwarded. You may use as a primer and a reverse primer.

  According to a particularly preferred embodiment of the present invention, the primer set used in the present invention includes two types of loop-forming primers as a forward primer and a reverse primer. That is, in this embodiment, the first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion. And a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) present 5 ′ to the sequence (A) in the target nucleic acid sequence is 5 ′ to the sequence (Ac ′). The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end. A sequence (D ′) that is hybridized to a complementary sequence (Dc) of a sequence (D) that is included in a portion and that is present 5 ′ to the sequence (C) in the target nucleic acid sequence is the sequence (Cc ′ ) On the 5 ′ side.

  According to another embodiment of the present invention, the primer set used in the present invention includes two kinds of folding primers as a forward primer and a reverse primer. That is, in this embodiment, the first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion. And a folded sequence (B-Bc ′) containing two nucleic acid sequences that hybridize to each other on the same strand is included on the 5 ′ side of the sequence (Ac ′), and is included in the primer set. The second primer comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and two hybridizing to each other A folded sequence (D-Dc ′) containing a nucleic acid sequence on the same strand is included on the 5 ′ side of the sequence (Cc ′).

  According to another preferred embodiment of the present invention, the primer set used in the present invention includes a combination of a loop-forming primer and a folding primer as a primer pair. That is, in this embodiment, the first primer included in the primer set according to the present invention includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence in the 3 ′ end portion. And a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) present 5 ′ to the sequence (A) in the target nucleic acid sequence is 5 of the sequence (Ac ′). 3) a sequence (Cc ′) that is hybridized to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence is a second primer included in the primer set. 'Folding sequence (D-Dc') containing two nucleic acid sequences, which are included in the terminal portion and hybridize to each other, on the same strand is included on the 5 'side of the sequence (Cc'). The

  A possible mechanism of action for the nucleic acid amplification reaction using the loop-forming primer (first primer) and the folding primer (second primer) will be described with reference to FIG. 3 (FIGS. 3a and 3b). In FIG. 3, for simplification of description, the two sequences to be hybridized are complementary to each other, but the present invention is not limited thereby. First, the first primer hybridizes to the sense strand of the target nucleic acid, and an extension reaction of the primer occurs (FIG. 3 (a)). Next, a stem-loop structure is formed on the extended strand (−), whereby a new first primer hybridizes to the sequence (A) on the target nucleic acid sense strand that has become a single strand (FIG. 3 (b) )), An extension reaction of the primer occurs, and the previously synthesized extension strand (-) is desorbed. Next, the second primer hybridizes to the sequence (C) on the released extended strand (−) (FIG. 3 (c)), the extension reaction of the primer occurs, and the extended strand (+) is synthesized. (FIG. 3 (d)). A stem-loop structure is formed at the 3 ′ end of the generated extended strand (+) and the 5 ′ end of the extended strand (−) (FIG. 3 (e)), and the free 3 ′ end of the extended strand (+) At the same time as the extension reaction occurs from the loop tip, the extended chain (-) is detached (FIG. 3 (f)). By the extension reaction from the loop tip, a hairpin-type double-stranded nucleic acid in which the extension strand (−) is bonded to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Bc) is generated, The first primer hybridizes to the sequence (A) and the sequence (Bc) (FIG. 3 (g)), and an extended chain (−) is generated by the extension reaction (FIG. 3 (h) and (i)). . In addition, a free 3 ′ end is provided by the folded sequence present at the 3 ′ end of the hairpin type double-stranded nucleic acid (FIG. 3 (h)), and by an extension reaction therefrom (FIG. 3 (i)), A single-stranded nucleic acid having a folded sequence at both ends and alternately containing an extended strand (+) and an extended strand (−) through the sequences derived from the first and second primers is generated (FIG. 3 (j )). In this single-stranded nucleic acid, since the free 3 ′ end (starting point of complementary strand synthesis) is provided by the folded sequence present at the 3 ′ end (FIG. 3 (k)), the same extension reaction is repeated, The chain length is doubled per extension reaction (FIGS. 3 (l) and (m)). In addition, in the extended strand (−) from the first primer desorbed in FIG. 3 (i), the free 3 ′ end (the complementary strand synthesis origin) is provided by the folded sequence present at the 3 ′ end. For this reason (FIG. 3 (n)), a stem-loop structure is formed at both ends by the extension reaction therefrom, and the extension strand (+) and the extension strand (-) are alternately included via the sequence derived from the primer. Double-stranded nucleic acid is generated (FIG. 3 (o)). In this single-stranded nucleic acid as well, the complementary strand synthesis starting point is sequentially provided by the loop formation at the 3 'end, so that extension reactions occur one after another. The single-stranded nucleic acid thus automatically extended contains sequences derived from the first primer and the second primer between the extended strand (+) and the extended strand (−). Therefore, it is possible for each primer to hybridize to cause an extension reaction, whereby the sense strand and the antisense strand of the target nucleic acid are significantly amplified.

  The primer set used in the present invention can include one or more third primers in addition to the first primer and the second primer. The third primer may be any one that hybridizes to the target nucleic acid sequence or its complementary sequence, but preferably does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. .

  In the present invention, “non-competing” means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.

  When the target nucleic acid is amplified by the folding primer, the amplification product has the target nucleic acid sequence and its complementary sequence alternately as illustrated in FIG. A folded sequence or loop structure exists at the 3 'end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided thereby. The third primer can be annealed to the target sequence present in the single-stranded portion when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis starting point is provided in the target nucleic acid sequence in the amplification product, and an extension reaction occurs therefrom, so that the nucleic acid amplification reaction is performed more rapidly.

  The third primer is not necessarily limited to one type, and two or more types of third primers may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers typically have a different sequence from the folded primer, but may hybridize to partially overlapping regions as long as they do not compete with these primers. The chain length of the third primer is preferably 2 to 100 nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.

  The third primer has an auxiliary function for proceeding the nucleic acid amplification reaction with the folded primer more rapidly. Accordingly, the third primer preferably has a Tm lower than the Tm of each 3 'end of the folded primer. Further, the amount of the third primer added to the amplification reaction solution is preferably smaller than the amount of each of the folded primers.

  Examples of the third primer include a primer having a structure capable of forming a loop as described in WO 02/24902 and giving a starting point for complementary strand synthesis to the loop portion. However, the present invention is not limited to this. That is, as long as it is within the target nucleic acid sequence, it may provide a complementary strand synthesis origin at any site.

  The primer set used in the present invention may further comprise an outer primer that hybridizes to a region 3 'from the target nucleic acid sequence on the template nucleic acid.

  The primers included in the primer set are composed of deoxyribonucleotides and / or ribonucleotides. In the present invention, “ribonucleotide” (sometimes simply referred to as “N”) refers to ribonucleoside triphosphate such as ATP, UTP, CTP, GTP and the like. Furthermore, ribonucleotides include these derivatives, for example, ribonucleotides (α-thio-ribonucleotides) in which the oxygen atom of the phosphate group at the α-position is replaced with a sulfur atom.

  The primer includes an oligonucleotide primer composed of unmodified deoxyribonucleotides and / or modified deoxyribonucleotides, and an oligonucleotide primer composed of unmodified ribonucleotides and / or modified ribonucleotides, unmodified deoxyribonucleotides and / or Also included are chimeric oligonucleotide primers containing modified deoxyribonucleotides and unmodified ribonucleotides and / or modified ribonucleotides.

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

  In the nucleic acid amplification method according to the present invention, a nucleic acid amplification reaction using the above-described primer set is performed using a nucleic acid molecule containing a target nucleic acid sequence as a template.

  The template nucleic acid containing the target nucleic acid sequence used in the nucleic acid amplification reaction may be either DNA or RNA. DNA includes any of cDNA, genomic DNA, and synthetic DNA. RNA includes all RNA, mRNA, rRNA, siRNA, hnRNA, and synthetic RNA. These nucleic acids can be prepared, for example, from blood, tissues, cells, biological samples such as animals and plants, or microorganism-derived samples separated from biological samples, food, soil, waste water, and the like.

  Isolation of the template nucleic acid can be performed by any method, and examples thereof include a method using a lysis treatment with a surfactant, sonication, shaking and stirring using glass beads, and a French press. Further, 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 template nucleic acid is a double-stranded nucleic acid such as genomic DNA or PCR fragment isolated by the above method, or a single strand such as cDNA prepared by reverse transcription from total RNA or mRNA. Any of the nucleic acids can be used. In the case of the above double-stranded nucleic acid, it can be used more optimally by carrying out a denaturing step 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 Reverse transcriptase 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. For the purpose of the present invention, an enzyme having reverse transcription activity at high temperature is optimal, and for example, DNA polymerase derived from Thermus bacteria (such as Tth DNA polymerase) or DNA polymerase derived from Bacillus bacteria can be used. Examples of particularly preferred enzymes include, for example, thermophilic Bacillus genus-derived DNA polymerases such as Bacillus stearothermophilus (hereinafter referred to as “B. st”) DNA polymerase (Bst DNA polymerase), and Bacillus stearothermophilus. Examples thereof include a DNA polymerase (Bca DNA polymerase) derived from cardiotenax (hereinafter referred to as “B. ca”), such as BcaBEST DNA polymerase and Bca (exo-) DNA polymerase. For example, Bca DNA polymerase does not require manganese ions in the reaction, and can synthesize cDNA while suppressing secondary structure formation of template RNA under high temperature conditions.

  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.

  In the present invention, the nucleic acid amplification reaction is performed under isothermal conditions. Here, “isothermal” refers to maintaining an approximately constant temperature condition such that the enzyme and the primer can substantially function. Furthermore, 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. To do. The nucleic acid amplification reaction under a constant temperature condition can be carried out by keeping the temperature at which the activity of the enzyme used can be maintained. In this 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 near or below the melting temperature (Tm) of the primer, The level of stringency is preferably set in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, more preferably about 35 ° C to about 65 ° C.

  The polymerase used in the nucleic acid amplification reaction is preferably one having strand displacement activity (strand displacement ability), and any of normal temperature, intermediate temperature, and heat resistance can be suitably used. Further, this polymerase may be either a natural body or a mutant having an artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that the DNA polymerase has substantially no 5 '→ 3' exonuclease activity. Examples of such DNA polymerase include B.I. st, B.B. Examples include mutants lacking the 5 'to 3' exonuclease activity of thermophilic Bacillus genus DNA polymerases such as ca, Klenow fragment of E. coli DNA polymerase I, and the like. Examples of the DNA polymerase used in the nucleic acid amplification reaction include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z -Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Thermonator DNA polymerase and the like.

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

  In the nucleic acid amplification reaction, in order to increase the amplification efficiency of the nucleic acid, a melting temperature adjusting agent can be added to the reaction solution. The melting temperature (Tm) of a nucleic acid is generally determined by the specific nucleotide sequence of the double stranded 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 such a melting temperature adjusting agent, it is possible to lower the melting temperature of the duplex forming part between two nucleic acids, in other words, to reduce the strength of the duplex formation. It becomes. 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 easier for the next primer to hybridize to the target region after the extension reaction by 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 influence the hybridization conditions, such as salt concentration, reaction temperature, etc. The Therefore, the melting temperature adjusting agent is not particularly limited, but is preferably dimethyl sulfoxide (DMSO), betaine, formamide or glycerol, or any combination thereof, more preferably dimethyl sulfoxide (DMSO). .

  Furthermore, in the nucleic acid amplification reaction, 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, and saccharides, and is not particularly limited.

  Furthermore, in the nucleic acid amplification reaction, a reagent for enhancing the heat resistance of an enzyme such as DNA polymerase or reverse transcriptase can be added to the reaction solution as an enzyme stabilizer. 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, but is preferably a saccharide, more preferably a mono- or oligosaccharide, more preferably trehalose, sorbitol or mannitol, or these It is set as the mixture of 2 or more types.

  The presence of the amplification product obtained by the nucleic acid amplification reaction can be detected by many various methods. One method is the detection of amplification products of a specific size by general gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green. As another method, it is possible to detect 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. As yet another method, there is a method using an immunochromatograph. In this method, it has been devised to use a chromatographic medium utilizing a label detectable with the naked eye (immunochromatography method). If the amplified fragment and the labeled probe are hybridized, and a capture probe capable of hybridizing with a further different sequence of the amplified fragment is fixed to the chromatographic medium, it can be trapped at the fixed part. Detection with is possible. As a result, simple detection can be performed with the naked eye. Furthermore, in the nucleic acid amplification method by the present inventors, the amplification efficiency in the nucleic acid amplification reaction is very high, so that the amplification product can also be detected indirectly by using the fact that pyrophosphate is generated as a byproduct of amplification. As such a method, for example, there is a method of visually observing the white turbidity of the reaction solution using the fact that pyrophosphoric acid binds to magnesium in the reaction solution to cause white precipitation of magnesium pyrophosphate. As another method, there is a method utilizing the fact that the concentration of magnesium ions in the reaction solution is remarkably reduced by forming an insoluble salt by strongly binding pyrophosphate to metal ions such as magnesium. In this method, a metal indicator whose color tone changes according to the magnesium ion concentration (for example, Eriochrome Black T, Hydroxy Naphthol Blue, etc.) is added to the reaction solution, and the color change of the reaction solution is visually observed. This makes it possible to detect the presence or absence of amplification. Furthermore, by using Calcein or the like, it is possible to visually observe the increase in fluorescence accompanying the amplification reaction, so that the amplification product can be detected in real time.

  The nucleic acid amplification method according to the present invention has a remarkably high sequence specificity. In particular, according to this nucleic acid amplification method, a nucleic acid having a sequence different from the target nucleic acid sequence is not amplified at all, and no amplification product is obtained even if the difference is one base. Such a high degree of sequence specificity according to the present invention is far beyond the expectation of those skilled in the art.

  The nucleic acid amplification method having high sequence specificity according to the present invention is suitable for determining the presence or absence of a mutation in a nucleic acid sequence in a nucleic acid sample. Therefore, according to the present invention, there is provided a method for determining the presence or absence of a mutation in a nucleic acid sequence in a nucleic acid sample by an isothermal nucleic acid amplification reaction, comprising: (a) preparing a nucleic acid sample; (b) recA protein (C) the primer set described above, wherein at least one primer contained in the primer set has at least one target sequence in the nucleic acid sample due to the presence or absence of the mutation. A step of preparing a primer set that is designed to cause a mismatch, and (d) a step of performing an isothermal nucleic acid amplification reaction with the primer set in the presence of the nucleic acid sample and the RecA protein, A method comprising is provided. As used herein, the term “mutation” encompasses any substitution, deletion and insertion of one or more nucleotides.

  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 multiple consecutive mismatches, mismatches caused by insertion and / or deletion of one or more bases, and combinations thereof.

  The one or more mismatches can be a single base mismatch, a plurality of consecutive mismatches, or a plurality of discontinuous mismatches.

  The above primers that cause mismatches due to the presence or absence of mutations are appropriately designed by those skilled in the art by comparing a target nucleic acid sequence having a mutation to be detected with a target nucleic acid sequence not having the mutation. can do. That is, the primer may be designed so as to hybridize to a region containing nucleotides different between these two target nucleic acid sequences. In this case, if the primer is designed to include a sequence complementary to the target nucleic acid sequence having a mutation, it will cause a mismatch due to the absence of the mutation, while complementary to the target nucleic acid sequence having no mutation. If it is designed to contain typical sequences, mismatches will be caused by the presence of mutations.

  If a loop-forming primer is used, this primer should be designed to produce one or more mismatches between the sequence (A) and the sequence (Ac ′) depending on the presence or absence of the mutation. Can do. Alternatively, this primer can also be designed to produce one or more mismatches between the sequence (Bc) and the sequence (B ′) due to the presence or absence of the mutation.

  When a folding primer is used, the primer may be designed to produce one or more mismatches between the sequence (C) and the sequence (Cc ′) depending on the presence or absence of the mutation. it can.

  When a third primer is used, this primer can be designed to cause one or more mismatches with the nucleic acid sequence or its complementary sequence due to the presence or absence of the mutation.

  Other conditions for the nucleic acid amplification reaction can be set in the same manner as in the nucleic acid amplification method according to the present invention. For example, in the nucleic acid amplification reaction, a polymerase having the above-described strand displacement ability is preferably used. Moreover, you may use the above-mentioned melting temperature adjusting agent, the above-mentioned enzyme stabilizer, etc. as needed.

  As a result of the mutation detection method according to the present invention, when an amplification product is obtained using a primer that causes a mismatch due to the presence of the mutation, it can be determined that the mutation does not exist in the nucleic acid sample. If no amplification product is obtained, it can be determined that the mutation is present. On the other hand, if an amplification product is obtained using a primer that causes a mismatch due to the absence of mutation, it can be determined that the mutation is present in the nucleic acid sample. Conversely, no amplification product is obtained. If it is, it can be determined that the mutation does not exist.

  In order to carry out the nucleic acid amplification method or the mutation detection method according to the present invention, necessary reagents can be combined into a kit. Therefore, according to the present invention, there is provided a kit for amplifying a target nucleic acid sequence in a template nucleic acid by an isothermal nucleic acid amplification reaction, comprising (a) RecA protein, and (b) the above-described primer set. A kit is provided. Furthermore, according to the present invention, there is provided a kit for determining the presence or absence of a mutation in a nucleic acid sequence in a nucleic acid sample by an isothermal nucleic acid amplification reaction, comprising: (a) a RecA protein; and (b) the primer set described above The at least one primer included in the primer set is designed to cause one or more mismatches with a target sequence in the nucleic acid sample due to the presence or absence of the mutation. A kit comprising a primer set is provided. The kit according to the present invention may further contain the above-mentioned reagents such as polymerase having strand displacement ability, dNTP, buffer solution, reaction vessel, instructions and the like.

  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.

Example 1: Amplification of a target nucleic acid sequence using a pair of loop-forming primers and RecA protein In this example, a pair of loop-forming primers and a RecA protein derived from Thermus thermophilus are used to produce EGFR (epidermal growth factor receptor) contained in the human genome. ) A nucleic acid amplification reaction was performed to amplify the target nucleic acid sequence in the gene.

  The positional relationship between the sequence around the target nucleic acid sequence in the human genome (SEQ ID NO: 1) and each primer is as shown in FIG. Specifically, primers having the following sequences were designed.

EGFR18F-01: 5′- CCCAGCACTT GAGGATCTTGAAGGAAACTG-3 ′ (SEQ ID NO: 2),
EGFR18R-06: 5'- CGGCACGGTG AGCCCAGAGGCCTGTGCCAG-3 '(SEQ ID NO: 3).

  For the forward primer EGFR18F-01, the sequence on the 3 'end side (20mer: other than the underlined portion) anneals to the template, and after the extension reaction, the sequence on the 5' end side (10mer: underlined portion) It is designed to hybridize to the region starting from 16 bases downstream of the 3 ′ terminal base of the primer on the extended strand. As for reverse primer EGFR18R-06, the sequence on the 3 'end side (20mer: other than the underlined portion) is annealed to the template, and after the extension reaction, the sequence on the 5' end side (10mer: underlined portion) is the primer. It is designed to hybridize to the region starting from 16 bases downstream of the 3 ′ terminal base of the primer on the extended strand.

Next, a reaction solution having the following composition: 10 × Buffer (Buffer: New England BioLabs attached to Bst DNA Polymerase), MgSO ₄ (4 mM), DMSO (5%), SYBR Green I (100,000 times the final concentration) Dilution, Molecular Probe), dNTP (0.4 mM each), forward primer EGFR18F-01 (3.2 μM), reverse primer EGFR18R-06 (3.2 μM), Human Genomic DNA (1.6 ng / μl, Promega), Bst DNA polymerase (8 U / μl, New England BioLabs), RecA protein (5 ng / μl), and ATP (1 mM) were prepared and incubated at 60 ° C. for 120 minutes. These reactions were performed using a real-time PCR apparatus Mx3000P (Stratagene).

  FIG. 5 shows changes with time in the amount of amplification product in the nucleic acid amplification reaction. In FIG. 5, squares indicate samples containing both the template (human genomic DNA) and RecA protein, circles indicate samples that contain the template and no RecA protein, and diamonds do not contain the template and contain RecA protein The triangles indicate samples that do not contain both the template and RecA protein.

  All amplification products for samples containing the template were confirmed to have the target nucleic acid sequence by sequencing. According to FIG. 5, although the amplification product was obtained in the sample containing the template (squares and circles), the sample further containing the RecA protein showed higher amplification efficiency (squares). In the sample containing no template, an amplification product was obtained in the sample not containing RecA protein (triangle), but no amplification product was obtained in the sample containing RecA protein (diamond).

Example 2: Effect of a single base mutation on amplification of a target nucleic acid sequence using a pair of loop forming primers and a RecA protein In this example, in order to confirm the specificity of the nucleic acid amplification reaction according to the present invention, a single base is included in the target nucleic acid sequence. A nucleic acid amplification reaction was performed using a loop-forming primer designed for a sequence having a mutation, and the result was compared with the result of a nucleic acid amplification reaction using a loop-forming primer for a sequence without a single nucleotide mutation.

  For this purpose, a pair of loop-forming primers was designed for each of a sequence containing a single nucleotide mutation and a sequence not containing this, targeting a region within the CYP2C19 * 2 gene contained in the human genome. In addition, an inner primer and an outer primer were also designed. The positional relationship between the sequence around the amplification region (SEQ ID NO: 4) in the CYP2C19 * 2 gene and each primer is as shown in FIG. Specifically, primers having the following sequences were designed.

(1) Loop-forming primer specific for wild-type sequence:
FIP-W: 5′- CCGGGAAATAAT CTTTTAATTTAATAAATTATTGTTTTCTCTTAG-3 ′ (SEQ ID NO: 5),
BIP-W: 5′- CGGGAACCC GTGTTCTTTTACTTTCTCC-3 ′ (SEQ ID NO: 6).

(2) Loop-forming primer specific for mutant sequence:
FIP-M: 5′-C T GGGAAATAATCTTTTAATTTAATAAATTATTGTTTTCTCTTAG-3 ′ (SEQ ID NO: 7),
BIP-M: 5′-C A GGAACCCGTGTTCTTTTACTTTCTCC-3 ′ (SEQ ID NO: 8).

  FIP-W and BIP-W are primer pairs specific for the wild type sequence. In FIP-W, the sequence at the 3 'end (33mer: other than the underlined portion) is annealed to the template, and after the extension reaction, the sequence at the 5' end (12mer: underlined) is extended by the primer. It is designed to hybridize to a region on the strand starting from 29 bases downstream of the 3 ′ terminal base of the primer. In BIP-W, the sequence at the 3 'end (19mer: other than the underlined portion) anneals to the template, and after the extension reaction, the sequence at the 5' end (9mer: underlined portion) is extended by the primer. It is designed to hybridize to a region on the strand starting from 47 bases downstream of the 3 ′ terminal base of the primer. On the other hand, FIP-M and BIP-M are primer pairs specific to the mutant sequence. These primers differ from FIP-W and BIP-W only at the second base (underlined) from the 5 'end corresponding to the single nucleotide mutation.

(3) Inner primer and outer primer:
LOOP-F: 5′-GATAGTGGGAAAATTATTGC-3 ′ (SEQ ID NO: 9),
LOOP-B: 5′-CAAATTACTTAAAAACCTTGCTT-3 ′ (SEQ ID NO: 10),
F3: 5′-CCAGAGCTTGGCATATTGTATC-3 ′ (SEQ ID NO: 11),
B3: 5′-AGGGTTGTTGATGTCCAT-3 ′ (SEQ ID NO: 12).

  LOOP-F and LOOP-B (inner primer), F3 (upstream outer primer) and B3 (downstream outer primer) consist only of sequences that hybridize to the template in the region shown in FIG.

Next, a reaction solution having the following composition: Tris-HCl (20 mM, pH 8.8), KCl (10 mM), (NH ₄ ) ₂ SO ₄ (10 mM), MgSO ₄ (4 mM), betaine (0.8 M), Triton X-100 (0.1%), dNTPs (0.4 mM each), a pair of loop-forming primers specific to wild type or mutant sequences (1600 nM each), inner primers LOOP-F and LOOP-B ( 800 nM), upstream outer primer F3 (200 nM), downstream outer primer B3 (200 nM), human genome with wild type sequence (40 ng), Bst DNA polymerase (8 U / μl, New England BioLabs), SYBR Green I (final concentration) 100,000-fold dilution, Molecular Probe), RecA protein (5 ng / μl), and A P containing (1 mM); was prepared and incubated which 60 minutes at 60 ° C.. These reactions were performed using a real-time PCR apparatus Mx3000P (Stratagene).

  FIG. 7 shows changes with time in the amount of amplification product in the nucleic acid amplification reaction. In FIG. 7, squares indicate samples containing primer sets specific to wild-type sequences, RecA and ATP, circles indicate samples containing primer sets specific to wild-type sequences, but no RecA and ATP. Shown, diamonds indicate samples containing primer sets specific for the mutant sequence, RecA and ATP, triangles indicate samples containing primer set specific for the mutant sequence but no RecA and ATP.

  All amplification products were confirmed to have the target nucleic acid sequence by sequencing. According to FIG. 7, when a loop-forming primer having no mismatch with the template was used, a large amount of amplification product was detected within 30 minutes regardless of the presence or absence of RecA protein (squares and circles). Even when a loop-forming primer having a mismatch with the template was used, an amplification product was detected between 25 and 50 minutes in the sample not containing RecA protein (triangle). On the other hand, when RecA protein was contained, no amplification product was detected by the loop-forming primer having a mismatch (diamonds). From these results, it was shown that the nucleic acid amplification method according to the present invention has remarkably high sequence specificity, and can identify, for example, a single-base mismatch in a template.

Example 3: Amplification of a target nucleic acid sequence using a pair of a loop-forming primer and a folding primer and a RecA protein In this example, a human genome is prepared using a primer pair consisting of a loop-forming primer and a folding primer and a RecA protein derived from T. thermophilus. A nucleic acid amplification reaction was performed to amplify the target nucleic acid sequence.

  The positional relationship between the sequence around the target nucleic acid sequence in the human genome (SEQ ID NO: 13) and each primer is as shown in FIG. Specifically, primers having the following nucleic acid sequences were designed.

Loop-forming primer:
2D6 * 44-F1: 5'- CGCTGCACAT GGCCTGGGGCCTCCTGCTCA-3 '(SEQ ID NO: 14)

  2D6 * 44-F1 is a primer specific for CYP2D6 * 1. In 2D6 * 44-F1, the sequence in the 3 'end region (20mer: other than the underlined portion) anneals to the template, and after the extension reaction, the sequence in the 5' end side (10mer: underlined portion) depends on the primer. It is designed to hybridize to a region starting from 16 bases downstream of the 3 ′ terminal base of the primer on the extended strand.

Folding primer:
2D6 * 44-SR2: 5'- TTTATATATATATAAA CCCCTGCACTGTTTCCCAGA-3 '(SEQ ID NO: 15)

  2D6 * 44-SR2 is a primer specific for both CYP2D6 * 1 and CYP2D6 * 44. In 2D6 * 44-SR2, the sequence on the 3 'end side (20mer) anneals to the template, and the sequence on the 5' end side (16mer: underlined) is folded within that region to take the structure shown in Fig. 2. Designed to be

Next, a reaction solution having the following composition: Tris-HCl (20 mM, pH 8.8), KCl (10 mM), (NH ₄ ) ₂ SO ₄ (10 mM), MgSO ₄ (5 mM), DMSO (5%), SYBRgreen (100,000 dilution, TaKaRa), Triton X-100 (0.1%), dNTP (0.4 mM), 2D6 * 44-F1 (5 μM), 2D6 * 44-SR2 (5 μM), human genome DNA (40 ng), Bst Polymerase (8 U / μL, New England Biolabs), RecA protein (10 ng / μL), and ATP (1.5 mM) were prepared and incubated for 90 minutes. These reactions were performed using a real-time PCR apparatus Mx3000p (Stratagene).

  FIG. 9 shows changes with time in the amount of amplification product in the nucleic acid amplification reaction. In FIG. 9, circles indicate samples containing a primer set specific to CYP2D6 * 1, squares indicate a primer set specific to CYP2D6 * 1, RecA and ATP-containing samples, and triangles indicate CYP2D6 * 1. Indicates a sample containing a primer set specific to, but no template DNA, and the diamond indicates a sample containing a primer set specific to CYP2D6 * 1, RecA and ATP, but no template DNA.

  All amplification products of samples containing the template were confirmed to contain the target nucleic acid sequence by sequencing. As is clear from FIG. 9, the addition of RecA and ATP promoted the amplification reaction as compared to the case of no addition. In addition, non-specific amplification could be suppressed by adding RecA and ATP in the sample without template.

FIG. 1 is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a loop-forming primer. FIG. 2 is a diagram illustrating the structure of a folding primer. FIG. 3a is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a loop-forming primer and a folding primer. FIG. 3b is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a loop-forming primer and a folding primer. FIG. 4 is a diagram showing a nucleotide sequence (SEQ ID NO: 1) around a target nucleic acid on an EGFR (epidermal growth factor receptor) gene contained in the human genome, and a region used for primer design. FIG. 5 is a graph showing the time course of nucleic acid amplification reaction using a pair of RecA protein and loop-forming primer. FIG. 6 is a diagram showing the nucleotide sequence (SEQ ID NO: 4) around the target nucleic acid on the CYP2C19 * 2 gene contained in the human genome and the region used for primer design. FIG. 7 is a graph showing the influence of a single base mismatch in a template in a nucleic acid amplification reaction using a pair of RecA protein and a loop-forming primer. FIG. 8 shows the nucleotide sequence (SEQ ID NO: 13) around the target nucleic acid on the CYP2D6 * 1 gene contained in the human genome, and the region used for primer design. FIG. 9 is a graph showing changes over time in a nucleic acid amplification reaction using RecA protein and a pair of a loop-forming primer and a folding primer.

Claims (52)

A method of amplifying a target nucleic acid sequence in a template nucleic acid by a nucleic acid amplification reaction under isothermal conditions,
(A) preparing a template nucleic acid containing a target nucleic acid sequence;
(B) preparing a RecA protein;
(C) preparing a primer set that enables amplification of the target nucleic acid sequence under isothermal conditions, and (d) a nucleic acid amplification reaction under isothermal conditions with the primer set in the presence of the template nucleic acid and RecA protein. A method comprising the steps of:   At least one kind of primer included in the primer set enables the 5 ′ end portion of the nucleic acid strand to hybridize on the same strand in the nucleic acid strand obtained by the extension reaction of the primer on the template. The method of claim 1, wherein:   At least one primer included in the primer set comprises a sequence (Ac ′) that hybridizes to a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and A sequence (B ′) that hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) is included on the 5 ′ side of the sequence (Ac ′). The method according to claim 1 or 2.   At least one primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. The method according to claim 1 or 2, comprising a folded sequence (B-Bc ') containing two nucleic acid sequences on the same strand, on the 5' side of the sequence (Ac '). The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The target nucleic acid sequence contains a sequence (D ′) that hybridizes to the complementary sequence (Dc) of the sequence (D) existing 5 ′ to the sequence (C) on the 5 ′ side of the sequence (Cc ′). The method according to claim 1 or 2, wherein The first primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. Comprising a folded sequence (B-Bc ′) containing two nucleic acid sequences on the same strand on the 5 ′ side of the sequence (Ac ′),
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 1 or 2, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). . The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 1 or 2, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). .   The method according to any one of claims 1 to 7, wherein the primer set comprises one or more third primers that hybridize to the target nucleic acid sequence or a complementary sequence thereof.   The method according to any one of claims 1 to 7, wherein the primer set comprises an outer primer that hybridizes to a region 3 'from the target nucleic acid sequence on a template nucleic acid.   The method according to any one of claims 1 to 7, wherein the nucleic acid amplification reaction is performed in the presence of ATP and / or an ATP analog.   The method according to any one of claims 1 to 7, wherein a polymerase having strand displacement ability is used.   The method according to any one of claims 1 to 7, wherein the nucleic acid amplification reaction is performed in the presence of a melting temperature adjusting agent.   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. A kit for amplifying a target nucleic acid sequence in a template nucleic acid by a nucleic acid amplification reaction under isothermal conditions,
A kit comprising (a) a RecA protein, and (b) a primer set that allows amplification of the target nucleic acid sequence under isothermal conditions.   At least one kind of primer included in the primer set enables the 5 ′ end portion of the nucleic acid strand to hybridize on the same strand in the nucleic acid strand obtained by the extension reaction of the primer on the template. 15. The kit according to claim 14, wherein   At least one primer included in the primer set comprises a sequence (Ac ′) that hybridizes to a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and A sequence (B ′) that hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) is included on the 5 ′ side of the sequence (Ac ′). The kit according to claim 14 or 15.   At least one primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. The kit according to claim 14 or 15, comprising a folded sequence (B-Bc ') containing two nucleic acid sequences on the same strand, on the 5' side of the sequence (Ac '). The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The target nucleic acid sequence contains a sequence (D ′) that hybridizes to the complementary sequence (Dc) of the sequence (D) existing 5 ′ to the sequence (C) on the 5 ′ side of the sequence (Cc ′). The kit according to claim 14 or 15, wherein The first primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. Comprising a folded sequence (B-Bc ′) containing two nucleic acid sequences on the same strand on the 5 ′ side of the sequence (Ac ′),
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The kit according to claim 14 or 15, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). . The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The kit according to claim 14 or 15, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). .   The kit according to any one of claims 14 to 20, wherein the primer set comprises one or more third primers that hybridize to the target nucleic acid sequence or a complementary sequence thereof.   The kit according to any one of claims 14 to 20, wherein the primer set comprises an outer primer that hybridizes to a region 3 'of the target nucleic acid sequence on a template nucleic acid.   21. A kit according to any one of claims 14 to 20, further comprising ATP and / or an ATP analog.   The kit according to any one of claims 14 to 20, further comprising a polymerase having strand displacement ability.   The kit according to any one of claims 14 to 20, further comprising a melting temperature adjusting agent.   The kit according to claim 25, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof. A method for determining the presence or absence of a mutation in a nucleic acid sequence in a nucleic acid sample by a nucleic acid amplification reaction under isothermal conditions,
(A) a step of preparing a nucleic acid sample;
(B) preparing a RecA protein;
(C) a primer set capable of isothermal amplification of a target nucleic acid sequence, wherein at least one primer contained in the primer set is a target sequence in the nucleic acid sample due to the presence or absence of the mutation A step of preparing a primer set, which is designed to produce one or more mismatches between, and (d) in the presence of the nucleic acid sample and the RecA protein under isothermal conditions by the primer set A method comprising a step of performing a nucleic acid amplification reaction.   At least one kind of primer included in the primer set enables the 5 ′ end portion of the nucleic acid strand to hybridize on the same strand in the nucleic acid strand obtained by the extension reaction of the primer on the template. 28. The method of claim 27, wherein:   At least one primer included in the primer set comprises a sequence (Ac ′) that hybridizes to a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and A sequence (B ′) that hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) is included on the 5 ′ side of the sequence (Ac ′). A method according to claim 27 or 28.   At least one primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. The method according to claim 27 or 28, comprising a folded sequence (B-Bc ') containing two nucleic acid sequences on the same strand on the 5' side of the sequence (Ac '). The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The target nucleic acid sequence contains a sequence (D ′) that hybridizes to the complementary sequence (Dc) of the sequence (D) existing 5 ′ to the sequence (C) on the 5 ′ side of the sequence (Cc ′). 29. The method of claim 27 or 28, wherein The first primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. Comprising a folded sequence (B-Bc ′) containing two nucleic acid sequences on the same strand on the 5 ′ side of the sequence (Ac ′),
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 27 or 28, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). . The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The method according to claim 27 or 28, comprising a folded sequence (D-Dc ') containing two nucleic acid sequences that hybridize to the same strand on the 5' side of the sequence (Cc '). .   34. The method according to any one of claims 27 to 33, wherein the primer set comprises one or more third primers that hybridize to the target nucleic acid sequence or its complementary sequence.   The method according to any one of claims 27 to 33, wherein the primer set comprises an outer primer that hybridizes to a region 3 'from the target nucleic acid sequence on a template nucleic acid.   34. The method according to any one of claims 27 to 33, wherein the nucleic acid amplification reaction is performed in the presence of ATP and / or an ATP analog.   The method according to any one of claims 27 to 33, wherein a polymerase having strand displacement ability is used.   The method according to any one of claims 27 to 33, wherein the nucleic acid amplification reaction is performed in the presence of a melting temperature adjusting agent.   40. The method of claim 38, wherein the melting temperature modifier is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof. A kit for determining the presence or absence of a mutation in a nucleic acid sequence in a nucleic acid sample by a nucleic acid amplification reaction under isothermal conditions,
(A) a RecA protein, and (b) a primer set that enables amplification of a target nucleic acid sequence under isothermal conditions, wherein at least one primer contained in the primer set depends on the presence or absence of the mutation. A kit comprising a primer set designed to produce one or more mismatches with a target sequence in the nucleic acid sample.   At least one kind of primer included in the primer set enables the 5 ′ end portion of the nucleic acid strand to hybridize on the same strand in the nucleic acid strand obtained by the extension reaction of the primer on the template. 41. A kit according to claim 40.   At least one primer included in the primer set comprises a sequence (Ac ′) that hybridizes to a sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and A sequence (B ′) that hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) is included on the 5 ′ side of the sequence (Ac ′). 42. A kit according to claim 40 or 41.   At least one primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. 42. The kit according to claim 40 or 41, comprising a folded sequence (B-Bc ′) containing two nucleic acid sequences on the same strand, on the 5 ′ side of the sequence (Ac ′). The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and The target nucleic acid sequence contains a sequence (D ′) that hybridizes to the complementary sequence (Dc) of the sequence (D) existing 5 ′ to the sequence (C) on the 5 ′ side of the sequence (Cc ′). 42. The kit according to claim 40 or 41, which comprises: The first primer included in the primer set includes a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion and hybridizes to each other. Comprising a folded sequence (B-Bc ′) containing two nucleic acid sequences on the same strand on the 5 ′ side of the sequence (Ac ′),
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and 42. The kit according to claim 40 or 41, comprising a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to the same strand on the 5 ′ side of the sequence (Cc ′). . The first primer included in the primer set comprises a sequence (Ac ′) that hybridizes to the sequence (A) of the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and The sequence (B ′) hybridizes to the complementary sequence (Bc) of the sequence (B) existing 5 ′ to the sequence (A) on the 5 ′ side of the sequence (Ac ′). ,
The second primer included in the primer set comprises a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence at the 3 ′ end portion, and 42. The kit according to claim 40 or 41, comprising a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to the same strand on the 5 ′ side of the sequence (Cc ′). .   47. The kit according to any one of claims 40 to 46, wherein the primer set comprises one or more third primers that hybridize to the target nucleic acid sequence or a complementary sequence thereof.   47. The kit according to any one of claims 40 to 46, wherein the primer set comprises an outer primer that hybridizes to a region 3 'from the target nucleic acid sequence on a template nucleic acid.   47. A kit according to any one of claims 40 to 46, further comprising ATP and / or an ATP analog.   The kit according to any one of claims 40 to 46, further comprising a polymerase having strand displacement ability.   47. The kit according to any one of claims 40 to 46, further comprising a melting temperature adjusting agent.   52. The kit according to claim 51, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.

Images