JP5618227B2 - Nucleic acid amplification method and gene mutation detection method - Google Patents

Description

  The present invention relates to a nucleic acid amplification method and a gene mutation detection method.

  Smart Amplification Process2 (SmartAmp2) is an isothermal nucleic acid amplification method using an asymmetric primer set with a turnback primer (TP) and a folding primer (FP) as main primers. For example, medical and molecular biology It is used in a wide range of fields (Non-Patent Documents 1-5). TP is a primer capable of forming a stem-loop structure having a sequence annealed to a target nucleic acid sequence and a sequence which can be annealed to a sequence extended from the target nucleic acid sequence on the 5 'side. FP is a primer having a sequence that anneals to a target nucleic acid sequence and a sequence that can form a folded structure inside the primer on the 5 'side. In addition to these, the primer set of SmartAmp2 can use a boost primer (BP) and two outer primers (OP1 and OP2). SmartAmp2 provides an analysis result quickly (for example, within 30 minutes), has high detection sensitivity (for example, 100 times that of conventional PCR), and can be amplified from several copies. Therefore, SmartAmp2 is preferably applicable to early diagnosis of diseases.

Nature Methods 4 (2007) 257-262. Clin. Cancer Res. 13 (2007) 4974-4983. BioTechniques 43 (2007) 479-484. Biologicals 36 (2008) 234-238. Clin. Chem. 10 (2009) 1373.

  In SmartAmp2, it is required to control the nucleic acid amplification rate. That is, in general gene detection, a reduction in detection time is required, but detection of a gene mutation such as a single nucleotide polymorphism requires higher detection accuracy than in a reduction in detection time.

  Accordingly, an object of the present invention is to provide a method for amplifying a nucleic acid by the SmartAmp2 method, and a method for amplifying a nucleic acid capable of controlling the amplification rate of the nucleic acid and a method for detecting a gene mutation using the same.

The nucleic acid amplification method of the present invention is a nucleic acid amplification method for amplifying a target nucleic acid sequence using a primer set comprising at least two kinds of primers,
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 the sequence in the target nucleic acid sequence A primer (TP) comprising a sequence (B ′) that hybridizes to a complementary sequence (Bc) of the sequence (B) existing 5 ′ from (A) on the 5 ′ side of the sequence (Ac ′);
The second primer included in the primer set includes a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence, and hybridizes to each other. A primer (FP) containing a folded sequence (D-Dc ′) containing two nucleic acid sequences to soybean on the same strand on the 5 ′ side of the sequence (Cc ′);
The amplification rate of the nucleic acid is controlled by adjusting at least one of a melting temperature and free energy in the folded sequence of the second primer.

  The gene mutation detection method of the present invention is a gene mutation detection method for detecting a gene mutation by a nucleic acid amplification method, wherein the nucleic acid amplification method is the nucleic acid amplification method of the present invention. .

  As a result of various studies, the present inventors have been able to control the amplification rate of nucleic acid by adjusting either or both of the melting temperature and free energy of the FP folding sequence. And found out that the present invention. Since primer sequences used in other isothermal nucleic acid amplification methods such as the LAMP method all depend on the target nucleic acid sequence, the degree of freedom in primer design is limited. On the other hand, since the FP folding sequence of the present invention does not depend on the target nucleic acid sequence, it can be designed freely and has a wide range of adjustment. This is also one of the great advantages of the present invention.

FIG. 1 is a schematic diagram showing the action mechanism of nucleic acid synthesis by the first primer in the nucleic acid amplification method of the present invention. FIG. 2 is a schematic diagram showing an example of a second primer in the nucleic acid amplification method of the present invention. FIG. 3A is a schematic diagram showing an example of a basic mechanism of the nucleic acid amplification method of the present invention. FIG. 3B is a schematic diagram showing an example of a basic mechanism of the nucleic acid amplification method of the present invention. 4a to 4n are schematic diagrams showing other examples of the mechanism of the nucleic acid amplification method of the present invention. FIG. 5A is a graph showing the melting temperature (Tm) of the FP folded array in the example of the present invention, and FIG. 5B shows the free energy (ΔG) of the FP folded array. It is a graph. FIGS. 6A to 6D are graphs showing the relationship between FP type, ratio, and reaction time in the examples. FIG. 7 is a photograph showing electrophoresis of the amplification product in the Example. FIG. 8 is a photograph of electrophoresis of the amplified product amplified using the RI-labeled primer in the above example. FIG. 9 is a diagram showing a target sequence and a primer sequence in the Example.

  In the nucleic acid amplification method of the present invention, the control may be to accelerate the amplification rate of the nucleic acid by lowering the melting temperature and increasing the free energy. In this case, for example, the melting temperature may be set to 50 ° C. or lower, and the free energy may be set to 0 kcal / mol or higher.

  In the nucleic acid amplification method of the present invention, the control may be to reduce the nucleic acid amplification rate by increasing the melting temperature and decreasing the free energy. In this case, for example, the melting temperature may be set to 50 ° C. or higher, and the free energy may be set to, for example, less than 0 kcal / mol, preferably −1 kcal / mol or less. Further, the background may be prevented from increasing by slowing down the amplification rate of the nucleic acid. If an increase in background is prevented, for example, it is possible to improve accuracy in detecting genetic mutations such as single nucleotide polymorphisms (SNPs).

  In the nucleic acid amplification method of the present invention, the nucleic acid amplification rate may be controlled by adjusting the ratio of the second primer in the primer set.

  In the method for detecting a gene mutation of the present invention, examples of the gene mutation include base insertion, deletion, substitution and addition. Moreover, it is preferable to use the method for detecting a genetic mutation of the present invention for detecting SNPs.

  Next, the present invention will be described in detail.

  The primer set in the present invention includes at least two kinds of primers. The first primer (TP) 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 in the 3 ′ end portion, and the target nucleic acid sequence The 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 second primer (FP) included in the primer set includes 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 a folded sequence (D-Dc ′) containing two nucleic acid sequences that hybridize to each other on the same strand is included on the 5 ′ side of the sequence (Cc ′).

  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 of 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 EFFrisch, 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 primers 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 all or part of the sequence of a nucleic acid molecule complementary to the target nucleic acid.

  The mechanism of the amplification reaction with the first 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 first 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 first primer may include 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 a 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, and thereby the stem in the 5 ′ end portion of the synthesized 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 first primer hybridizes to this portion (FIG. 1 (b)). Thereafter, an extension reaction from the newly hybridized first 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 amplification reaction described above, 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 first 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 strand 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 carrying out the reaction, the 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 first primer in a 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 is formed by forming a stem-loop structure at the 5 ′ end of the synthesized complementary strand. The part of the sequence (A) on the nucleic acid 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 for a new primer to efficiently anneal to a 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 first primer according to a preferred embodiment of the present invention is the (X−) in the case where no intervening sequence exists between the sequence (Ac ′) and the sequence (B ′) constituting the primer. 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, more preferably 30 or more, and preferably 50 or less, more preferably 48 or less, and further preferably 42 or less.

  Further, when there is an intervening sequence (base number Y ′) between the sequence (Ac ′) and the sequence (B ′) constituting the primer, the first primer according to a 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 first primer has a chain length that allows base pairing with a target nucleic acid while maintaining the required specificity under given conditions. The primer has a chain length of preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. Moreover, 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').

  As described above, the second primer (FP) included in the primer set according to the present invention is 3 ′ of the complementary sequence of the target nucleic acid sequence (the strand opposite to the strand to which the first primer hybridizes). A folded sequence (D-Dc ′) containing a sequence (Cc ′) hybridizing to the sequence (C) of the terminal portion at the 3 ′ end portion and two nucleic acid sequences hybridizing to each other on the same strand It is included on the 5 ′ side of the sequence (Cc ′). The structure of such a second primer is, for example, as shown in FIG. 2, but is not limited to the sequence or the number of nucleotides shown in FIG. The length of the sequence (Cc ′) constituting the second primer is preferably 5 to 50 nucleotides, more preferably 10 to 30 nucleotides. The length of the folded sequence (D-Dc ′) 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 (D-Dc ′) 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 (Cc ′) and the folded sequence (D-Dc ′).

  In the present invention, the nucleic acid amplification rate is controlled by adjusting at least one of the melting temperature Tm and the free energy of the folded sequence of the second primer. When the melting temperature is low and the free energy is high, the folded array has an unstable structure. On the other hand, when the melting temperature is high and the free energy is low, the folded array has a stable structure. Here, the unstable structure means that the folded structure is easily broken, and the stable structure means that the folded structure is hardly broken. In the present invention, the melting temperature and free energy may be actual measured values or predicted values. The actual measured value and predicted value of the melting temperature and free energy can be determined by a conventionally known method.

  The melting temperature and free energy can be adjusted by, for example, the number of base pairs formed by hydrogen bonding and the GC content in the folded sequence. In the case of an unstable folded sequence, the number of base pairs is, for example, in the range of 2 to 100, preferably in the range of 4 to 60, and more preferably in the range of 6 to 40. In the case of an unstable folded sequence, the GC content is, for example, in the range of 0 to 80%, preferably in the range of 0 to 50%, more preferably in the range of 0 to 30%. In the case of an unstable folded arrangement, the melting temperature is, for example, 50 ° C. or lower, preferably 40 ° C. or lower, more preferably 30 ° C. or lower. In this case, the lower limit of the melting temperature is not particularly limited, but is, for example, −200 ° C. or higher, preferably −100 ° C. or higher. In the case of an unstable folded sequence, the free energy is, for example, 0 kcal / mol or more, preferably 1 kcal / mol or more, more preferably 2 kcal / mol or more. In this case, the upper limit of the free energy is not particularly limited, but is, for example, 100 kcal / mol or less. In the case of a stable folded sequence, the number of base pairs is, for example, in the range of 10 to 500, preferably in the range of 10 to 100, and more preferably in the range of 10 to 40. In the case of a stable folded sequence, the GC content is, for example, in the range of 20 to 100%, preferably in the range of 30 to 90%, and more preferably in the range of 40 to 80%. In the case of a stable folded arrangement, the melting temperature is, for example, 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 70 ° C. or higher. In this case, the upper limit of the melting temperature is not particularly limited, but is, for example, 100 ° C. or less. In the case of a stable folded sequence, the free energy is, for example, less than 0 kcal / mol, preferably −1 kcal / mol or less, more preferably −2 kcal / mol or less, particularly preferably −3 kcal / mol or less. In this case, the lower limit of the free energy is not particularly limited, but is, for example, −200 kcal / mol or more.

  A possible mechanism of action for the nucleic acid amplification reaction using the first primer and the 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 the primer extension reaction occurs (FIG. 3 (a)). Next, a stem-loop structure is formed on the extended strand (−), and thereby 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)), the extension reaction of the primer occurs, and the previously synthesized extended 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 extended strand (+) which is the free 3 ′ end. At the same time, an extension reaction occurs from the loop tip of the above, and the extension 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)), stem-loop structures are 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. Single-stranded nucleic acid is produced (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 elongation reactions occur one after another. In such a single-stranded nucleic acid that is automatically extended, sequences derived from the first primer and the second primer are contained between the extended strand (+) and the extended strand (−). Therefore, it is possible for each primer to hybridize to cause an extension reaction, and thereby the sense strand and the antisense strand of the target nucleic acid are significantly amplified.

  The primer set according to the present invention may include a third primer (boost primer: BP) in addition to the first primer and the second primer. The third primer hybridizes to the target nucleic acid sequence or its complementary sequence, and 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 first primer and the second primer, as described above, the amplification product has the target nucleic acid sequence and its complementary sequence alternately. A folded sequence or a 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 takes place 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 comprise a different sequence from the first primer and the second 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 mainly serves as an auxiliary function for proceeding the nucleic acid amplification reaction by the first primer and the second primer more rapidly. Therefore, the third primer preferably has a Tm lower than the Tm of each 3 'end of the first primer and the second primer. Moreover, it is preferable that the addition amount of the third primer to the amplification reaction solution is smaller than the addition amounts of the first primer and the second primer.

  Examples of the third primer include a primer having a structure capable of forming a loop as described in WO 02/24902 pamphlet 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.

  4A to 4N are schematic diagrams of the amplification reaction when the third primer (BP) is used. The amplification reaction includes (a) (b) (e) (f) (i) (l), (a) (b) (f) (j) (m), (a) (b) ( f) (j) (g) (k) (n), (a) (c) (g) (k) (n) or (a) (d) (h) Exists. Hereinafter, these routes will be described in order. First, a first route having the main routes of (a), (b), (e), (f), (i), and (l) will be described. In this route, first, the first primer (TP) hybridizes to the sense strand of the target nucleic acid ((a) in the figure), the extension reaction of the first primer (TP) occurs, and the extended strand tp_1 (Figure (b)). Next, a third primer (BP) is hybridized to the extended strand tp_1 ((b) in the same figure), and the extension reaction of the third primer (BP) occurs, resulting in an extended chain elBP (the same figure). (e)). In addition, the second primer (FP) hybridizes to the extended strand tp_1 (FIG. (B)), the extension reaction of the second primer (FP) occurs, and the extended strand fp_tp_1 is generated (FIG. ( f)). Next, the extended chain elBP is hybridized to the extended chain fp_tp_1 (FIG. (I)), and an extension reaction of the extended chain elBP occurs to generate an extended chain bp_fp_1 (FIG. (L)). Furthermore, a stem-loop structure is formed at the 3 'end of the extended strand bp_fp_1 ((1) in the figure), and an extension reaction occurs using the 3' end as a complementary strand synthesis origin. Next, a second route having the main routes of (a), (b), (f), (j), and (m) will be described. This route is common to the first route until the extended chain fp_tp_1 is generated ((f) in the figure). A stem-loop structure is formed at the 3 ′ end of the extended chain fp_tp_1 (figure (f)), and an extension reaction takes place with the 3 ′ end as the starting point for complementary chain synthesis, resulting in an extended chain fp_fp_2 (figure (j) )). Next, a stem-loop structure is formed at the 3 ′ end of the extended strand fp_fp_2 ((j) in the figure), and an extension reaction occurs using the 3 ′ end as a complementary strand synthesis origin to generate an extended strand fp_fp_4 (same as above). Figure (m)). Furthermore, a stem-loop structure is formed at the 3 'end of the extended strand fp_fp_4 ((m) in the figure), and an extension reaction occurs using the 3' end as a complementary strand synthesis origin. Next, a third route having the main routes of (a), (b), (f), (j), (g), (k), and (n) will be described. This route is common to the second route until the extended chain fp_fp_2 is generated ((j) in the figure). A first primer (TP) is hybridized to the extended chain fp_fp_2 (FIG. (J)), and an extension reaction of the first primer (TP) occurs to generate an extended chain tp_fp_1 (FIG. (G) ). A stem-loop structure is formed at the 3 ′ end of the extended chain tp_fp_1 (FIG. (G)), and an extension reaction occurs using the 3 ′ end as a complementary chain synthesis origin to generate an extended chain tp_tp_2 (FIG. (K)). )). In addition, a stem-loop structure is formed at the 3 ′ end of the extended chain tp_tp_2 ((k) in the figure), and an extension reaction takes place with the 3 ′ end as a complementary chain synthesis origin, resulting in an extended chain tp_tp_4 (in the same figure (n)). Furthermore, a stem-loop structure is formed at the 3 'end of the extended strand tp_tp_4 ((n) in the figure), and an extension reaction occurs using the 3' end as a complementary strand synthesis origin. In addition, the first primer (TP) is hybridized to the extended chain tp_tp_4 ((n) in the figure), and the extension reaction of the first primer (TP) occurs. Next, a fourth route having the main routes of (a), (c), (g), (k), and (n) will be described. In this pathway, first, the second primer (FP) hybridizes to the antisense strand of the target nucleic acid ((a) in the figure), and the extension reaction of the second primer (FP) occurs, and the extended strand fp_1 (Fig. (C)). Next, the first primer (TP) is hybridized to the extended chain fp_1 ((c) in the same figure), and the extension reaction of the first primer (TP) occurs to generate an extended chain tp_fp_1 (the same figure). (g)). After FIG. 5G, the third route is common. Finally, a fifth route having the main routes of (a), (d), and (h) in the figure will be described. In this pathway, first, the third primer (BP) hybridizes to the antisense strand of the target nucleic acid ((a) in the figure), and the extension reaction of the third primer (BP) occurs, and the extended strand bp_1 (Fig. (D)). Next, the first primer (TP) is hybridized to the extended strand bp_1 (FIG. (D)), the extension reaction of the first primer (TP) occurs, and the extended strand elTP is generated (FIG. (h)). The above-described reaction path is an exemplification, and the present invention is not limited or limited by the reaction path.

  The primers included in the primer set according to the present invention are composed of deoxynucleotides and / or ribonucleotides. In the present invention, “ribonucleotide” (sometimes simply referred to as “N”) refers to ribonucleotide triphosphate such as ATP, UTP, CTP, and GTP. 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 deoxynucleotides and / or modified deoxynucleotides, and an oligonucleotide primer composed of unmodified ribonucleotides and / or modified ribonucleotides, unmodified deoxynucleotides and / or Alternatively, chimeric oligonucleotide primers containing modified deoxynucleotides and unmodified ribonucleotides and / or modified ribonucleotides are also included.

  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 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.).

  The template nucleic acid or nucleic acid sample 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 of 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 or nucleic acid sample 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, a French press, and the like. In addition, when an endogenous nuclease is present, it is preferable to purify the isolated nucleic acid. Nucleic acid purification can be performed, for example, by phenol extraction, chromatography, ion exchange, gel electrophoresis, density-dependent centrifugation, and the like.

  More specifically, the template nucleic acid or the nucleic acid sample is a double-stranded nucleic acid such as genomic DNA or PCR fragment isolated by the above method, or a cDNA prepared by reverse transcription from total RNA or mRNA. 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 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 (RAV-2 RTase), Moloney murine leukemia virus-derived reverse transcriptase (MMLV RTase), and other reverse transcriptases. 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 a high temperature is optimal. For example, a DNA polymerase derived from Thermus sp. (Tth DNA polymerase, etc.), a DNA polymerase derived from Bacillus sp. As a particularly preferable enzyme, for example, as a DNA polymerase derived from a thermophilic Bacillus bacterium, B. st-derived DNA polymerase (Bst DNA polymerase); Ca-derived DNA polymerase (Bca DNA polymerase), for example, BcaBEST DNA polymerase, Bca (exo-) DNA polymerase, and the like can be mentioned. For example, Bca DNA polymerase does not require manganese ions for 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.

The polymerase used in the nucleic acid amplification reaction only needs to have a 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 with artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that this DNA polymerase has substantially no 5 ′ → 3 ′ exonuclease activity. Examples of such a DNA polymerase include thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as “B.st”) and Bacillus caldotenax (hereinafter referred to as “B.ca”). Examples include mutants lacking the 5 ′ → 3 ′ exonuclease activity of the derived DNA polymerase, Klenow fragment of DNA polymerase I derived from E. coli . 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, Therminater DNA polymerase and the like.

  Furthermore, in the nucleic acid amplification reaction, 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 as a template. The DNA polymerase reaction can be carried out with one kind of polymerase. Moreover, you may use combining DNA polymerase and above-mentioned reverse transcriptases, such as MMLV reverse transcriptase.

  Other reagents used in the nucleic acid amplification reaction include, for example, catalysts such as magnesium chloride, magnesium acetate, magnesium sulfate, substrates such as dNTP mix, Tris-HCl buffer, tricine buffer, sodium phosphate buffer, potassium phosphate buffer, etc. 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, a melting temperature adjusting agent can be added to the reaction solution in order to increase the amplification efficiency of the nucleic acid. 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, the melting temperature of the duplex forming part between two nucleic acids can be lowered, in other words, the strength of the duplex formation can be lowered. 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 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 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, saccharides, etc., 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 nucleic acid amplification reaction using the primer set according to the present invention can be performed isothermally. Therefore, according to a preferred embodiment of the present invention, the nucleic acid amplification reaction comprises the steps of preparing a nucleic acid amplification solution comprising a template nucleic acid or nucleic acid sample and the primer set according to the present invention, and the nucleic acid amplification solution. Incubating isothermally. 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 certain 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.

  In the nucleic acid amplification reaction, the amplification reaction is repeated until the enzyme is deactivated or one of the reagents including the primer is used up.

  In the above nucleic acid amplification reaction, a nucleic acid containing an unnatural nucleotide can be used as a template nucleic acid. As used herein, “non-natural nucleotide” means a nucleotide that contains a base other than the base (adenine, guanine, cytosine, and thymine or uracil) contained in a natural nucleotide, and that can be incorporated into a nucleic acid sequence. Examples thereof include xanthosines, diaminopyrimidines, isoG and isoC (Proc. Natl. Acad. Sci. USA 92, 6329-6333, 1995). In general, a nucleic acid amplification enzyme having no heat resistance is used for amplification of a target nucleic acid containing a non-natural nucleotide. On the other hand, since the nucleic acid amplification reaction can be performed at an isothermal temperature of, for example, about 50 ° C., the possibility that the nucleic acid amplification enzyme (DNA polymerase or the like) is inactivated is low as compared with the conventional PCR method. Therefore, the nucleic acid amplification reaction using the primer set according to the present invention is also effective for amplification of a target nucleic acid containing a non-natural nucleotide in which a nucleic acid amplification enzyme having no heat resistance is used. The enzyme used for the amplification of the nucleic acid containing the non-natural nucleotide is not particularly limited as long as it can amplify such a target nucleic acid, but Y188L / E478Q mutant HIV I reverse transcription particularly from the viewpoint of incorporation efficiency Enzymes, AMV reverse transcriptase, Klenow fragment of DNA polymerase, 9 ° N DNA polymerase, HotTub DNA polymerase and the like are suitable (Michael Sismour1 et al., Biochemistry 42, No. 28, 8598, 2003 / US Pat. No. 6,617,106). Description, Michael J. Lutz et al., Bioorganic & Medical Chemistry letters 8, 1149-1152, 1998, etc.). Furthermore, a substance that improves the heat resistance of the nucleic acid amplification enzyme, such as trehalose, can also be added to the reaction solution, whereby the target nucleic acid containing unnatural nucleotides can be more efficiently amplified.

  Next, examples of the present invention will be described. However, the present invention is not limited or limited by the following examples.

Prediction of melting temperature and minimum free energy of secondary structure. Tm value prediction is described in Primer 3 software package (S. Rozen et al., Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, 365-386 (2000). ) Using the “oligotm” software. Prediction of the minimum free energy of the secondary structure was performed using the “mfold” program (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi). In the simulation for predicting the Tm value and the minimum free energy, the ion conditions were set to 10 mmol / L Na ⁺ and 8 mmol / L Mg ²⁺ , and the temperature was set to 60 ° C.

2 types of FP folding array
FP self-priming occurs after denaturation and hairpin structure formation. Therefore, in order to analyze the relationship between the Tm value of the FP folded sequence and the hairpin structure stability based on the minimum free energy of the secondary structure and nucleic acid amplification by the SmartAmp2 method, the FP folded sequence was designed as follows. The Tm value of the FP folded array was changed within the range of 19.5 to 61.8 ° C., and the minimum free energy of the secondary structure was changed within the range of −4.3 to 1.6 kcal / mol. Furthermore, according to the Tm value and the minimum free energy of the secondary structure, the FP folded array was classified into two types, S-type and W-type. The W type has a low melting temperature and high free energy, and the S type has a high melting temperature and low free energy. FIG. 5A shows the Tm values of W1 and W2 which are two W-type FP folding arrays designed as described above, and the Tm values of S1 and S2 which are two S-type FP folding arrays. Show. FIG. 4B shows the minimum free energy of the secondary structure of W1 and W2, which are two W-type FP folding arrays designed as described above, and two S-type FP folding arrays, S1 and The minimum free energy of the secondary structure of S2 is shown.

The primer sequence for detecting the target gene detection primer sequences B2AR and UCP1 is determined using the published primer sequencing system (http://smap.gsc.riken.jp/smap/PrimerDesign.do). I went. FIG. 9A shows the sequences of UCP1 and B2AR around the target nucleic acid sequence and various detection primers. The upper part of the figure (A) (1) shows the sequence of UCP1 around the target nucleic acid sequence. The underline attached to the UCP1 sequence is a sequence to which various detection primers hybridize. The site shown in bold in the UCP1 sequence is a site where single nucleotide polymorphisms occur. The sequence of the UCP1 detection primer is shown in the lower part of FIG. Note that the sequences of UCP1 FP tail-W1 and UCP1 FP tail-S1 that hybridize to UCP1 are the same. In the upper part of FIGS. (A) and (2), the B2AR sequence around the target nucleic acid sequence is shown. The underline attached to the B2AR sequence is a sequence to which various detection primers hybridize. Further, the site shown in bold in the B2AR sequence is a site where a single nucleotide polymorphism occurs. The sequence of the B2AR detection primer is shown in the lower part of FIGS. Note that the sequences in which B2AR FP tail-W1 and B2AR FP tail-S1 hybridize to B2AR are identical.

Preparation of template plasmid A plasmid prepared by the following method was used as a template for the SmartAmp2 method so that genomic fragments other than the amplified region would not affect the results of the SmartAmp2 method. First, the region surrounding the target nucleic acid sequences of UCP1 and B2AR was amplified by PCR. The amplification product was inserted into the pGEM-T plasmid and cloned to obtain a plasmid containing UCP1 and B2AR target regions. The plasmid was used as a template for the SmartAmp2 method for amplifying UCP1 and B2AR. The plasmid was stored at −20 ° C. until used as a template for the SmartAmp2 method.

SmartAmp2 Method Nature Methods 4 (2007) 257-262, Clin Cancer Res. 13 (2007) 4974-4983. And Clin. Chem. 10 (2009) 1373. Using the method reported in, the SmartAmp2 method was performed as follows. The template plasmid used was diluted to 2000 copies / μL, then heated to 98 ° C. and denatured. The composition of the SmartAmp2 reaction solution is as shown below. The concentration of FP was 0.5, 1, 2, 4 or 8 μmol / L as shown in FIG. The reaction solution was prepared on ice to make 25 μL. By incubating the reaction solution at 60 ° C. for 120 minutes, a SmartAmp2 reaction was performed to amplify the nucleic acid. The generation of the amplification product was monitored every minute using a real-time fluorescence detection apparatus (trade name Mx3000P, manufactured by Stratagene).

(SmartAmp2 reaction solution)
20 mmol / L Tris-HCl buffer (pH 8.0)
10 mmol / L Potassium chloride 10 mmol / L Ammonium sulfate 8 mmol / L Magnesium sulfate 5% Dimethyl sulfoxide (DMSO)
0.1% Tween20
1.4mmol / L dNTP
2.0μmol / L TP
2.0μmol / L BP
0.5, 1, 2, 4 or 8 μmol / L FP
0.001% original SYBRGreen I
(Molecular Probe)
6units AacDNA polymerase
(Danafoam)
2000 copies template plasmid

SmartAmp2 reaction time determination method
The time from the start of the SmartAmp2 reaction to the maximum rate of change in fluorescence intensity monitored at each time point by the real-time fluorescence detector was defined as the SmartAmp2 reaction time. The time at which the fluorescence intensity change rate is maximized was determined by the following method. First, the difference in fluorescence intensity between adjacent time points was calculated every other minute, and the amount of change in fluorescence intensity at each time point was determined. Next, calculate the difference in average fluorescence intensity at 5 time points 2 minutes before, 1 minute before the reference time point, 1 hour after the reference time, 2 minutes after the reference time, and compare these values. The time period during which the amount of change in fluorescence intensity monotonously was determined. The reference time point at which the average value of the fluorescence intensity change amount was maximum was determined as the reaction time.

Gel electrophoresis
The amplification product of SmartAmp2 was analyzed by electrophoresis using 10% polyacrylamide gel (AMRESCO, Ohio, USA). An orange ruler (trade name) 100 bp DNA ladder (manufactured by Fermentas) was used as an electrophoresis size marker. The electrophoresis was performed at 35 W for 150 minutes in 1 × TBE buffer (40 mmol / L Tris, 20 mmol / L boric acid, 1 mmol / L EDTA). After the electrophoresis, the gel was stained with ethidium bromide solution (5 μg / ml) for 20 minutes, washed with deionized water for 20 minutes, and observed by UV transillumination.

³² P-ATP Labeling and ³² P-ATP by labeling autoradiography various primer of the primer was carried out in the manner described below. By reacting ³² P-ATP and T4 polynucleotide kinase (NEB, USA) with 37 P at 30 ° C. for 30 minutes in 50 μl of 1 × kinase buffer (NEB, USA), the above various primers were treated with ³² P-ATP. Subjected to phosphorylation. Next, the reaction was stopped by treatment at 65 ° C. for 20 minutes to obtain a labeled primer. The labeled primer was added to the primer mix used for the SmartAmp2 reaction solution to prepare the SmartAmp2 reaction solution. The SmartAmp2 reaction solution was incubated at 60 ° C. for 70 minutes to perform a SmartAmp2 reaction. The amplification product of the SmartAmp2 reaction was subjected to gel electrophoresis by the method described above. After the gel electrophoresis, the gel was analyzed with an image analyzer (manufactured by Fuji Film) to detect ³² P-ATP signal.

Influence of FP Fold Sequence on Nucleic Acid Amplification Rate in SmartAmp2 Method In order to investigate the relationship between the ratio of FP in the primer set and the minimum free energy of Tm value and secondary structure in the FP fold sequence and the amplification rate of the nucleic acid The following experiment was conducted. SmartAmp2 assays were performed on several targets including UCP1 and B2AR, with FP concentrations of 0.5, 1, 2, 4, 8 μmol / L. 6A to 6D illustrate UCP1 FP (UCP1-W) having a W1-type folded array, UCP1 FP (UCP1-S) having an S1-type folded array, and B2AR FP (B2AR) having a W1-type folded array, respectively. It is a graph which shows the relationship between the density | concentration of FP, and reaction time when performing SmartAmp2 method using B2AR FP (B2AR-S) which has -W) and S1 type | mold folding sequence | arrangement. In each graph, the horizontal axis represents the FP concentration (μmol / L), and the vertical axis represents the SmartAmp2 reaction time (minutes). As shown in FIGS. 4A and 4C, in the case of the FP having the W1-type FP folding sequence, the SmartAmp2 reaction time was constant regardless of the FP concentration. On the other hand, as shown in FIGS. 4B and 4D, in the case of the FP having the S1-type FP folding sequence, the higher the FP concentration, the longer the SmartAmp2 reaction time. Although data are not shown, in the case of target genes other than UCP1 and B2AR, a relationship depending on W1 type and S1 type of the FP folding sequence as described above was observed between the FP concentration and the SmartAmp2 reaction time. . Further, the above relationship was not observed with respect to the Tm value of the portion other than the folded array and the minimum free energy of the secondary structure. From the above results, it was found that the amplification rate of the nucleic acid can be controlled by increasing the ratio of the FP primer in the primer set. Furthermore, by decreasing the melting temperature and increasing the free energy, the amplification rate of the nucleic acid is accelerated, and by increasing the melting temperature and decreasing the free energy, It was found that the amplification rate was reduced.

Electrophoretic analysis of amplification products
Optimization of the FP primer is important not only for the amplification rate but also for the generation pattern of the amplification product. Therefore, in order to examine the relationship between the amount of FP primer added and the amplification product generation pattern, and the relationship between the FP primer folding sequence and the amplification product generation pattern, the B2AR SmartAmp2 amplification product was subjected to electrophoretic analysis. FIG. 7 shows the results of electrophoretic analysis of B2AR SmartAmp2 amplification products using B2AR FP (B2AR-W) having a W1-type folded sequence and B2AR FP (B2AR-S) having a S1-type folded sequence. As shown in the figure, the electrophoresis pattern of the amplification product was clearly different between B2AR-W and B2AR-S. Moreover, as shown in the figure, in B2AR-W and B2AR-S, the increase and decrease of signals in many bands were observed simultaneously according to the change in the FP concentration. These results indicate that there was a change in the nucleic acid amplification pathway by the SmartAmp2 method.

Autoradiographic analysis of amplification products labeled with RI
To examine the relationship between the amount of FP primer added and the amplification product generation pattern, the contribution of each of TP, BP, or FP in nucleic acid amplification, and the relationship between the FP primer folding sequence and the amplification product generation pattern, The B2AR SmartAmp2 amplification product obtained by amplifying BP or FP with ³² P-ATP label was analyzed by autoradiography. FIGS. 8A and 8B show B2AR SmartAmp2 amplification products using B2AR FP having a W1-type folded sequence (B2AR-W) and B2AR FP having a S1-type folded sequence (B2AR-S). The results of autoradiography analysis are shown respectively. In addition, from the left in the figure (A) and (B), when TP is labeled with ³² P-ATP (TP label), when BP is labeled with ³² P-ATP (BP label) and when FP is labeled with ³² P-ATP The analysis results for the case (FP label) are shown. Furthermore, for each reaction, the FP concentration was 0.5, 2 or 8 μmol / L. As shown in the results of the TP label and BP label in FIGS. 6A and 6B, the migration pattern of the amplification product containing TP or BP shows that the FP folding sequence is W1 type (B2AR-W) and S1 type. The case (B2AR-S) was clearly different. It was also found that the amount of amplification product containing TP or BP increased and the length increased as the FP concentration increased. This tendency was the same whether the FP folding array was the W1 type or the S1 type. FP contributes to the production of amplification products containing TP or BP by producing templates for amplification reactions. From this result, it was found that the amplification product containing TP or BP can be increased depending on the concentration of FP regardless of whether the FP folding sequence is of W1 type or S1 type. In addition, as shown in the FP label results in FIGS. 4A and 4B, the migration pattern of the amplification product containing FP is clearly different depending on whether the FP folding sequence is the W1 type or the S1 type. I understood. As shown in the results of the FP label in FIG. 6A, when the FP folding sequence was W1 type, the amount of amplification product containing FP was small. On the other hand, as shown in the result of the FP label in FIG. 5B, when the FP folding sequence was S1 type, there were relatively many amplification products containing FP. From this result, it was found that the amplification product containing FP can be increased by making the FP folding sequence S1 type. It was also found that amplification products containing FP increased depending on the concentration of FP. From the above results, it was found that the amplification product containing FP can be increased by changing the FP folding sequence to S1 type and decreasing the nucleic acid amplification rate. Such an inverse correlation between the nucleic acid amplification rate and the amount of amplification product containing FP was unexpected.

  The present invention relates to a nucleic acid sequence amplification method and a gene mutation detection method. According to the nucleic acid sequence amplification method of the present invention, the nucleic acid amplification rate can be controlled. On the other hand, in the case of general gene detection, it is required to shorten the detection time. However, detection of a gene mutation such as a single nucleotide polymorphism requires higher detection accuracy than the shortening of the detection time. For this reason, the nucleic acid sequence amplification method and gene mutation detection method of the present invention can be said to be extremely useful tools in the field of genetic engineering, for example.

Claims (9)

A nucleic acid amplification method for amplifying a target nucleic acid sequence using a primer set comprising at least two kinds of primers,
The first primer included in the primer set 3 'sequence at the end portion (A) sequence that hybridizes to (Ac' of the target nucleic acid sequence) the 3 'includes a distal portion, or One sequence (B') A primer containing 5 ′ side of the sequence (Ac ′),
Here, the sequence (B ′) is a sequence that hybridizes to the sequence (Bc),
The sequence (Bc) is a sequence existing on a complementary sequence of the target nucleic acid sequence, and
The sequence (Bc) is a sequence complementary to the sequence (B),
The sequence (B) is a sequence present 5 ′ to the target nucleic acid from the sequence (A),
The second primer included in the primer set includes a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence, and hybridizes to each other. A primer comprising a folded sequence (D-Dc ′) comprising two nucleic acid sequences for soybean on the same strand on the 5 ′ side of the sequence (Cc ′);
A method for amplifying a nucleic acid, wherein the nucleic acid amplification rate is accelerated by lowering the melting temperature of the folded sequence of the second primer and increasing the free energy . The melting temperature was set to 50 ° C. or less, and the method for amplifying a nucleic acid of claim 1, wherein for setting the free energy than 0 kcal / mol. A nucleic acid amplification method for amplifying a target nucleic acid sequence using a primer set comprising at least two kinds of primers,
The first primer included in the primer set 3 'sequence at the end portion (A) sequence that hybridizes to (Ac' of the target nucleic acid sequence) the 3 'includes a distal portion, or One sequence (B') A primer containing 5 ′ side of the sequence (Ac ′),
Here, the sequence (B ′) is a sequence that hybridizes to the sequence (Bc),
The sequence (Bc) is a sequence existing on a complementary sequence of the target nucleic acid sequence, and
The sequence (Bc) is a sequence complementary to the sequence (B),
The sequence (B) is a sequence present 5 ′ to the target nucleic acid from the sequence (A),
The second primer included in the primer set includes a sequence (Cc ′) that hybridizes to the sequence (C) of the 3 ′ end portion of the complementary sequence of the target nucleic acid sequence, and hybridizes to each other. A primer comprising a folded sequence (D-Dc ′) comprising two nucleic acid sequences for soybean on the same strand on the 5 ′ side of the sequence (Cc ′);
A method for amplifying a nucleic acid, wherein the nucleic acid amplification rate is reduced by increasing the melting temperature of the folded sequence of the second primer and decreasing the free energy . The nucleic acid amplification method according to claim 3, wherein the melting temperature is set to 50 ° C. or higher and the free energy is set to −1 kcal / mol or lower. The method for amplifying a nucleic acid according to claim 3 or 4, wherein an increase in background is prevented by reducing the amplification rate of the nucleic acid. Furthermore, the nucleic acid amplification method according to any one of claims 1 to 5 , wherein the nucleic acid amplification rate is controlled by adjusting a ratio of the second primer in the primer set. A method for detecting a gene mutation by detecting a gene mutation by a nucleic acid amplification method, wherein the nucleic acid amplification method is the nucleic acid amplification method according to any one of claims 1 to 6. Detection method of gene mutation. The method for detecting a gene mutation according to claim 7 , wherein the gene mutation is at least one of base insertion, deletion, substitution and addition. The method for detecting a gene mutation according to claim 7 , wherein the gene mutation is a single nucleotide polymorphism.