JP2001309785A - Method for detecting differences between gene sequences - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a difference between gene sequences. A method for detecting a difference in a base sequence between a first polynucleotide chain and a second polynucleotide chain which are mutually homologous. Using a novel nucleotide amplification method, loop-mediated amplification, in the presence of a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the first polynucleotide chain. , First
Wherein the polynucleotide chain and the second polynucleotide chain are used as templates to perform a nucleotide chain amplification reaction, respectively, and the amplified product is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a difference between gene sequences.

[0002]

2. Description of the Related Art With the rapid progress of genetic analysis technology in recent years and the increase in genetic information, techniques for diagnosing and testing diseases and constitutions of patients at the genetic level are becoming widespread. In particular, recently, a large number of single nucleotide polymorphisms (SNPs: single nucleotide p
olymorphism) has been mapped on the chromosome, and for some SNPs, the effects on protein function have already been identified. Under such circumstances, it is expected that it will be important to examine the types of SNPs of patients before drug administration in order to provide optimal medical care for each patient in clinical practice. To do so, each SN
A technique for accurately and rapidly analyzing the base at the Ps site is required. On the other hand, such an analysis technique exerts tremendous power also in the diagnosis of congenital diseases and acquired diseases caused by point mutations. For example, one of the congenital genetic diseases caused by point mutation is sickle cell disease. This disease
As shown in FIG. 1, one base in the hemoglobin gene is innately substituted from A to T, so that one glutamic acid constituting the hemoglobin protein is replaced with valine, and the mutation results in the three-dimensional structure of hemoglobin. It changes drastically and causes sickle cell disease. In addition, cancer is a typical example of an acquired genetic disease caused by a point mutation. For example, colorectal cancer develops in a large intestine polyp generated in the colonic mucosal epithelium through several stages of genetic mutation as shown in FIG. Above all, mutations in the p53 gene are often point mutations. Detection of such a point mutation can be effectively used for determining the risk of onset, the degree of malignancy of the disease, and the prognosis.

Conventionally, as a method for detecting SNPs or point mutations, PCR has been performed using primers designed to contain a sequence complementary to the SNPs to be detected or a sequence containing a point mutation site. How to do was known. However, in PCR, if complementary strand synthesis is erroneously performed, the product functions as a template for the subsequent reaction, and may cause erroneous results. In practice, only a single base difference at the end of the primer
It is said that it is difficult to precisely control PCR, and it is necessary to improve the specificity of a primer for a nucleotide chain.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for accurately and highly sensitively detecting a difference between gene sequences using a loop mediated amplification (LAMP) method.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by using a novel nucleotide chain amplification method, a loop-mediated amplification method, the present inventors have found that the internucleotide chain The difference was detected accurately and with high sensitivity, and the present invention was completed.

That is, the present invention relates to a method for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain which are homologous to each other, comprising the following steps: The first arbitrary sequence F1c, the second arbitrary sequence F2c and the third arbitrary sequence F2c from the 3 ′ end of the target region on the polynucleotide chain toward the 3 ′ end of the polynucleotide chain.
Are selected, and the fourth arbitrary sequence R1, the fifth arbitrary sequence R2, and the sixth arbitrary sequence R3 are sequentially arranged from the 5 ′ end of the target region toward the 5 ′ end of the polynucleotide chain. A step of selecting each, (B) the F
A sequence F2 complementary to 2c and the F2
A primer containing a sequence F3c complementary to the F3c, a primer containing a sequence F3 complementary to the F3c, a sequence identical to the R2 and a sequence R complementary to the R1 on the 5 ′ side of the sequence.
(C) preparing a primer containing 1c and a primer containing the same sequence as R3;
A first polynucleotide chain and a second polynucleotide in the presence of a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the polynucleotide chain, the primer and the polymerase; The above-mentioned detection method comprising a step of performing a nucleotide chain synthesis reaction using each of the nucleotide chains as a template, and (D) a step of detecting the obtained amplification product.

Further, the present invention provides a method for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain, which are homologous to each other, comprising the following steps: The first arbitrary sequence F1c, the second arbitrary sequence F2c and the third arbitrary sequence F2c from the 3 ′ end of the target region on the polynucleotide chain toward the 3 ′ end of the polynucleotide chain.
Selecting each of the fourth arbitrary sequence R1 and the fifth arbitrary sequence R3 in order from the 5 ′ end of the target region toward the 5 ′ end of the polynucleotide chain, (b) A) a sequence F2 complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2, and a primer containing the same sequence as the R1 and a primer containing the same sequence as the R3, respectively. (C) preparing a first nucleotide sequence in the presence of a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the first polynucleotide chain, the primer and a polymerase; Performing a nucleotide chain synthesis reaction using each of the polynucleotide chain and the second polynucleotide chain as templates, and (d) obtaining the resulting amplified product. Which is the detection method comprising the step of detecting.

Furthermore, the present invention is the above-mentioned detection method, wherein the nucleotide chain analog is a peptide nucleic acid. further,
The present invention is the above-mentioned detection method, wherein the nucleotide chain analog comprises 8 to 30 bases.

Further, the present invention relates to F1c, F2c, F3
The region of c, R1, R2, R3, or F2c to R2 is the first region.
The above detection method comprises a difference in base sequence to be detected between the polynucleotide chain of (a) and the second polynucleotide chain. Furthermore, the present invention is the above detection method, wherein the nucleotide chain synthesis reaction is performed using a strand displacement polymerase.

Furthermore, the present invention is the above-mentioned detection method, wherein the nucleotide chain synthesis reaction is carried out in the presence of a melting temperature adjusting agent. Furthermore, the present invention is the above-mentioned detection method, wherein the melting temperature adjusting agent is betaine, dimethyl sulfoxide, formamide, or a tetraalkylammonium salt. Further, the present invention provides a betaine of 0.2 to 3M.
The above detection method is a concentration of

Furthermore, the present invention is the above-mentioned detection method, wherein the difference in base sequence is caused by a point mutation. Furthermore, the present invention is the above-mentioned detection method, wherein the difference in the nucleotide sequence is caused by a single nucleotide polymorphism. Furthermore, the present invention is a kit for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain, which are homologous to each other, using a nucleotide chain amplification reaction, It is a kit containing the elements of

(I) a nucleotide chain analog having a base sequence complementary to a base containing a base at a different site to be detected in the first polynucleotide chain;
The first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third arbitrary sequence F from the 3 ′ end of the target region on the polynucleotide chain toward the 3 ′ end of the polynucleotide chain.
3c, and a fourth optional sequence R1, a fifth optional sequence R2, and a sixth optional sequence R3, respectively, are sequentially selected from the 5 'end of the target amplification region toward the 5' end of the polynucleotide chain. In this case, a sequence F2 complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2, a sequence F3 complementary to the F3c
A primer comprising the same sequence as that of R2,
A primer containing a sequence R1c complementary to the R1 on the 5 ′ side, and a primer containing the same sequence as the R3;
(III) a polymerase that catalyzes a strand displacement type complementary strand synthesis reaction, and (IV) a nucleotide that serves as a substrate for the element (III).

Further, the present invention provides a kit for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain, which are homologous to each other, by utilizing a nucleotide chain amplification reaction. And a kit comprising the following components: (I) a nucleotide chain analog having a base sequence complementary to a base containing a base at a different site to be detected in the first polynucleotide chain, (II) a target region 3 on the first polynucleotide chain The first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third arbitrary sequence F3c are respectively selected from the 'end toward the 3' end of the polynucleotide chain, and the polynucleotide is selected from the 5 'end of the target amplification region. A fourth arbitrary sequence R1 in order toward the 5 'end of the nucleotide chain;
And when the fifth arbitrary sequence R3 is selected, a sequence F2 complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2, a sequence F3 complementary to the F3c A primer containing the same sequence as R1 and a primer containing the same sequence as R3. (III) a polymerase that catalyzes a strand displacement type complementary strand synthesis reaction, and (IV) a substrate of element (III). Nucleotides.

Further, the present invention is the above kit, wherein the nucleotide chain analog is a peptide nucleic acid. Furthermore, the present invention is the above-mentioned detection method, wherein the nucleotide chain analog comprises 8 to 30 bases.

Further, the present invention relates to F1c, F2c, F3
The region of c, R1, R2, R3, or F2c to R2 is the first region.
The kit comprising a nucleotide sequence to be detected which is different between the polynucleotide chain of the above and the second polynucleotide chain. Further, the present invention is the aforementioned kit, further comprising a detecting agent for detecting a product of the nucleic acid synthesis reaction.

Further, the present invention relates to a method for detecting the presence of
The kit is a probe comprising the same nucleotide sequence as the region between the two or the complementary strand thereof. Further, the present invention provides
The above kit wherein the probe is labeled with a particle. Hereinafter, the present invention will be described in detail.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The method for detecting a difference between gene sequences according to the present invention comprises the steps of:
A method for detecting a difference in base sequence between a polynucleotide chain of a first polynucleotide chain and a base sequence complementary to a sequence containing a base at a different site to be detected in a first polynucleotide chain First in the presence of nucleotide chain analog
Using the polynucleotide chain and the second polynucleotide chain as templates, respectively, performing a nucleotide chain amplification reaction by a loop-mediated amplification method (hereinafter, also referred to as a LAMP method), and detecting an amplification product. is there.
Here, the “first polynucleotide chain” refers to a control nucleotide chain having a reference base sequence for detecting a difference in base sequence between sequences. The “second polynucleotide chain” refers to a nucleotide to be tested for checking whether or not a certain portion on the nucleotide chain is the same base as the first polynucleotide chain with respect to the first polynucleotide chain. Refers to a chain. "Different site to be detected"
In the first polynucleotide chain and the second polynucleotide chain, it refers to a difference between base sequences to be detected. "Complementary" refers to both cases in which it is completely complementary and in which it can be annealed under stringent conditions.

1. Method for detecting differences between gene sequences of the present invention (1) Loop-mediated amplification method (LAMP method) The method for detecting differences between gene sequences of the present invention is characterized by utilizing the LAMP method. One embodiment of the LAMP method will be described below with reference to FIGS. That is,
In the present amplification method, first, in the template polynucleotide chain including the target region (A in FIG. 3), the first arbitrary sequence F1c, the first arbitrary sequence F1c from the 3 ′ end of the target region toward the 3 ′ end of the polynucleotide chain. The second arbitrary sequence F2c and the third arbitrary sequence F3c are respectively selected, and similarly, the fourth arbitrary sequence R1, the fifth arbitrary sequence R2, and the sixth arbitrary sequence F5c from the 5 ′ end to the 5 ′ end of the target region. An arbitrary sequence R3 is selected (B in FIG. 3). Next, a primer F2c complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2 (hereinafter, also referred to as FA primer) are annealed to the F2c sequence on the template polynucleotide chain,
DNA strand synthesis is started from F2 of the FA primer as a starting point (C in FIG. 3). Here, "annealing" means that a nucleotide chain forms a double-stranded structure by base pairing based on Watson-Crick's law. Next, a primer containing the sequence F3 complementary to F3c (hereinafter, also referred to as F3 primer) is annealed to the F3c sequence of the template polynucleotide chain (FIG. 3, D). Strand displacement DNA starting from the annealed F3 primer
Chain synthesis is performed (E in FIG. 3). By the above synthesis reaction, the sequence “(3 ′) F3c-
Sequence complementary to “F2c-F1c-target region-R1-R2-R3 (5 ′)” “(5 ′) F3-F2-F1-target region-R1c
-R2c-R3c (3 ') "(FIG. 3,
F) and “(5 ′) F having the same sequence as F1c at the 5 ′ end.
1c-F2-F1-target region-R1c-R2c-R3c (3 ') "
Is obtained (FIG. 3, G). Here, the sequence “(5 ′) F1c-F2-F1-target region-R1c
-R2c-R3c (3 ') "forms a hairpin loop by intrachain hydrogen bonding between the sequences F1c and F1 present on the 5' side (FIG. 3, G). Then, the sequence “(5 ′) F1c-F2-F1-target region-R1c-R2c-R
3c (3 ′) ”, annealed to the R2c sequence, a primer containing the R2 sequence and a sequence R1c complementary to the R1 sequence on the 5 ′ side of the sequence (hereinafter also referred to as RA primer),
DNA chain synthesis is started using the annealed RA primer as a synthesis starting point (H in FIG. 3). Then, the sequence "(5 ')
F1c-F2-F1-target region-R1c-R2c-R3c
A primer containing the same sequence as R3 (hereinafter also referred to as R3 primer) is annealed to the R3c sequence of the nucleotide chain containing (3 ′) ”(FIG. 3, I). Annealed R
Starting from the three primers, strand displacement DNA synthesis is performed (J in FIG. 4). By the above synthesis reaction, the sequence “(5 ′) F1c-F2-F1-target region-R1c-R2c-R3
c (3 ′) ”, a complementary nucleotide chain“ (3 ′) F1-
F2c-F1c-target region-R1-R2-R3 (5 ′) ”(FIG. 4, K) and“ (3 ′) F1-F2c-F1-target having F1 at the 3 ′ end and R1c at the 5 ′ end. Region-R1-R2-R1c
(5 ′) ”(FIG. 4, L). The sequence “(3 ′) F1-
F2c-F1c-target region-R1-R2-R1c (5 ′) ”
A hairpin loop is formed by intrachain hydrogen bonding between the sequences F1 and F1c present on the 3 ′ side and between the sequences R1 and R1c present on the 5 ′ side (FIG. 4, L). Next, the sequence “(3 ′) F1-F2c-F1c-target region-R1-R2-R1
The FA primer is annealed to F2c in the hairpin loop portion on the 3 ′ side of “c (5 ′)” (FIG. 4, M). Next, DNA strand synthesis is started with F2 of the FA primer as a starting point, while DNA strand synthesis is started with F1 annealed by intrachain hydrogen bonding as a synthesis starting point (FIG. 4, M).
As a result, the single-stranded overhanging structure "-target sequence-R1c-R
2c-R1 "is obtained (FIG. 4,
N). This structure is composed of R1c and R1
A hairpin loop is formed by forming an intra-chain hydrogen bond between and (Fig. 4, N). Next, R1 annealed by the intrachain hydrogen bond is used as a synthesis starting point.
Initiate DNA strand synthesis (FIG. 4, N). By the above synthesis reaction, the sequence “(3 ′) R1-R2-R1c-target region-F1
-F2-F1c-target sequence-R1-R2c-R1c-target sequence-
F1-F2c-F1c-target sequence-R1-R2-R1c (5 ') "
(FIG. 4, O) and a sequence having F1c at the 3 ′ end and R1 at the 5 ′ end “(3 ′) F1c-F2-F1-target region-R1c-R
2c-R1 (5 ′) ”is obtained (FIG. 4, P). Both of the above two sequences have R2c and F2 due to intrachain hydrogen bonding.
A hairpin loop is formed with 2 and R2c as loop portions. An RA primer anneals to the RA portion forming a hairpin in the two sequences, DNA synthesis starting from the primer starts, and synthesis of a nucleotide chain including the target sequence proceeds exponentially. Become.

Further, another embodiment of the loop-mediated amplification method is shown in FIGS. That is, in this embodiment,
First, a template polynucleotide chain containing a target region (in FIG. 5,
a) selecting a first optional sequence F1c, a second optional sequence F2c and a third optional sequence F3c from the 3 ′ end of the target region above toward the 3 ′ end of the polynucleotide chain; A fourth arbitrary sequence R1 and a fifth arbitrary sequence R3 are respectively selected from the 5 ′ end to the 5 ′ end of the target region (b in FIG. 5). Next, a sequence F2 complementary to the F2c and a FA primer containing the same sequence as the F1c on the 5 ′ side of the F2 are annealed to the F2c sequence on the template polynucleotide chain, and the F2 of the FA primer is used as a starting point. To initiate DNA synthesis (in FIG. 5,
c). Next, an F3 primer containing a sequence F3 complementary to the sequence is annealed to F3c of the template polynucleotide (FIG. 5, d), and a strand displacement type DNA strand synthesis is performed using the annealed F3 primer as a synthesis starting point. (Figure 5
Medium, e). By the above synthesis reaction, the sequence “(3 ′) F3c-F2c-F1c-target region-
R1-R3 (5 ') "and a complementary nucleotide chain"(5') F3-F2-F1-target region-R1c-R3c (3 ') "
(F) at the 5 ′ end and “(5 ′) F1c-F2-F1-target region-R1
A polynucleotide chain containing "c-R3c (3 ')" is obtained (FIG. 5, g). Here, the sequence “(5 ′) F1c-F2-F
“1-Target region-R1c-R3c (3 ′)” forms a hairpin loop by intrachain hydrogen bonding between the sequences F1c and F1 present on the 5 ′ side (FIG. 5, g). Then, the sequence "(5 ')
F1c-F2-F1-target region-R1c-R2c-R3c
(3 ′) ”, an R1 primer having the same sequence as R1 and an R3 primer having the same sequence as R3 are annealed to the sequences R1c and R3c, respectively (in FIG. 6,
h). Next, while DNA strand synthesis is performed from the R1 primer, the strand displacement type D
The NA chain is synthesized (FIG. 6, h). By the above synthesis reaction, the sequence “(5 ′) F1c-F2-F1-target region-R1c-
R3c (3 ') ", the complementary nucleotide chain"(3') F
1-F2c-F1c-target region-R1-R3 (5 ′) ”(FIG. 6,
i) and “(3 ′) F1-F2c-F having F1 at the 3 ′ end.
1-target region-R1 (5 ′) ”is obtained (FIG. 6, j). The sequence “(3 ′) F1-F2c-F1c-target region-R1
(5 ′) ”forms a hairpin loop by intrachain hydrogen bonding between the sequences F1 and F1c present on the 3 ′ side (FIG. 6,
j). Next, F2c is added to F2c of the hairpin loop portion of the sequence “(3 ′) F1-F2c-F1c-target region-R1 (5 ′)”.
Anneal the A primer (FIG. 6, k). Then
DNA strand synthesis is started from F2 of the FA primer as a starting point, while F annealed by intrachain hydrogen bonding.
The DNA strand synthesis is started using No. 1 as a synthesis starting point (FIG. 6,
k). As a result, the single-stranded overhang structure “-target sequence-R1
A double-stranded DNA having "c" is obtained (FIG. 6, l). Next, an R1 primer is annealed to R1c of the single-stranded protruding structure portion, and DNA strand synthesis is started using the primer as a synthesis starting point (FIG. 6, l). By the above synthesis reaction, a nucleotide chain "(3 ') R1c-target region-complementary to the sequence"(5') R1-target region-F1c-F2c-F1-target sequence-R1c (3 ') ". F1-F2-F1c-target sequence-R
1 (5 ′) ”(FIG. 6, m) and“ (5 ′) F1c-F2-F1-target region-R1c (3 ′) ”having F1c at the 5 ′ end (FIG. 6, n) . In the above sequence, the R1 primer anneals to the R1c portion, DNA synthesis starting from the primer starts, and then the synthesis of a nucleotide chain containing the target sequence proceeds exponentially.

The FA used in the above amplification reaction
Primers and RA primers as inner primers,
Although the F3 primer and the R3 primer are also called outer primers, nucleotide chain synthesis from the outer primer needs to be started after nucleotide chain synthesis from the inner primer as described in the above description. As a method for achieving this, there is a method in which the concentration of the inner primer is set higher than the concentration of the outer primer. Specifically, the concentration of the inner primer is 2 to 50 times higher than the concentration of the outer primer.
It can be carried out by setting it higher by a factor of 2, preferably 4 to 10 times.

Since PCR conventionally used for amplifying a nucleotide chain involves a temperature cycle, it is said that the temperature change stress decreases the enzyme activity due to a long decrease in the enzymatic activity, thereby lowering the efficiency of synthesizing a base sequence. In a preferred embodiment using the LAMP method, no temperature cycle is required in the nucleotide chain amplification step, so that synthesis can be reliably achieved even with a long base sequence. The template polynucleotide chain, arbitrary sequence, primers and the like used in the above amplification method will be described in detail below.

(2) Template Polynucleotide Chain The template nucleotide chain in the LAMP method (1) (that is, the nucleotide chain targeted for the detection method of the present invention) includes DNA and RNA. In addition, nucleotide chains into which artificial nucleotide derivatives have been incorporated, and those that have been chemically modified after nucleotide chain formation, etc., may be used as the template in the above (1) as long as they function as templates for complementary strand synthesis. it can. Examples of the source of the nucleotide chain include a biological source and a chemical source. Here, examples of the biological source include biological individuals such as animals, plants, microorganisms, mycoplasmas, and viruses, biological cells such as cultured cells, excretions of organisms, and extracts thereof, and chemical sources. Examples include chemical compounds. Also, the nucleotide chain may be derived from an isolate from the source. Examples of the derivative include a cDNA synthesized using mRNA as a template, and a product amplified using a nucleotide chain derived from a biological source as a template.

(3) Selection of Each Arbitrary Sequence In one embodiment of the LAMP method (1), first, the arbitrary sequences F1c, F2c, F3c, R
It is necessary to select 1, R2 and R3, etc. In making the selection, it is preferable to make a selection that meets the following selection criteria. First, the amplification product obtained by the LAMP method is not intermolecular annealing but F F as shown in FIG.
It is preferable to select so that intramolecular annealing occurs preferentially between the 1c sequence and the F1 sequence and between the R1 sequence and the R1c sequence to form a terminal loop structure. For example, in order to cause intramolecular annealing to occur preferentially, in selecting an arbitrary sequence, it is important to consider the distance between the F1c and F2c sequences and the distance between the R1 and R1c sequences. . Specifically, it is preferable that both sequences are selected so as to exist at a distance of 0 to 500 bases, preferably 0 to 100 bases, most preferably 10 to 70 bases. Here, the numerical values indicate the number of bases that do not include the F1c and F2c sequences themselves and the R1 and R2 sequences themselves, respectively. In addition, in order to preferentially cause intramolecular annealing, in selecting an arbitrary sequence, it is also effective to consider the melting temperature of a duplex formed between each arbitrary sequence and the arbitrary sequence. . Here, "Melting temperature (T
m) "means 50% of the nucleic acids with complementary nucleotide sequences
Refers to the temperature at which is brought into a base-paired state. In particular,
The melting temperature of the inner primer is set higher than the melting temperature of the outer primer. That is, (F3-F3
melting temperature between C sequences and between R3-R3c sequences) ≦ (F2-
Melting temperature between F2c sequences and between R2-R2c sequences) ≦
(Melting temperature between F1-F1c sequence and between R1-R1c sequence) so that F1c, F2c, F3c, R1, R
2, and R3. Here, (melting temperature between F2-F2c regions and between R2-R2c regions) ≦ (F1-F1c
The melting temperature between the regions and between the R1-R1c regions) was defined as the F2 sequence of the FA primer or the R2 sequence of the RA primer.
F1-F before the two sequences anneal to the loop
This is for annealing between the 1c regions or between the R1 and R1c regions. Annealing between the F1-F1c regions or between the R1-R1c regions is an intramolecular reaction, and may proceed preferentially. The melting temperature of the double strand formed by the nucleotide chain and the complementary strand of the nucleotide chain,
It can be theoretically calculated based on the combination of the length of the double strand and the base constituting the base pairing. For example,
The melting temperature Tm under normal salt concentration (0.18 M NaCl) can be calculated by the following formula [J. Marmur and P.
Doty: J. Mol. Biol., 5: 109 (1962)].

[0024]

[Equation 1] Tm = 69.3 + 0.41 (% G + C)

The distance between F2c and R2 is not particularly limited, as long as the distance allows complementary strand synthesis. The distance at which the complementary strand can be synthesized also depends on the synthesis ability of the DNA polymerase, but under suitable conditions, synthesis can be performed sufficiently even with a length of about 1 kbp. More specifically, when Bst DNA polymerase is used, the distance between F2c and R2 is 800 b
p, preferably less than 500 bp, is reliably synthesized. It should be noted that the same selection criteria apply to other embodiments of the LAMP method illustrated by FIGS.

(4) Design of Primers for LAMP Method Based on each arbitrary sequence selected in the above (3), primers essential for the LAMP method are designed as follows. As shown in FIG. 3, the FA primer is a primer having a sequence F2 complementary to an arbitrary sequence F2c and a sequence identical to the F1c sequence on the 5 ′ side of the F2. The F3 primer is a primer having a sequence complementary to the arbitrary sequence F3c. The RA primer is a primer having the same sequence as R2 and a sequence R1c complementary to R1 on the 5 ′ side of the sequence. The R3 primer is a primer containing the same sequence as R3. In designing the FA primer and RA primer, the amplification product obtained by the LAMP method
As in (L), intramolecular annealing occurs preferentially between the F1c and F1 sequences and between the R1 and R1c sequences,
It is necessary to design to form a terminal loop structure. For that purpose, the distance between the F1c sequence and the F2 sequence and the distance between the R1c sequence and the R2 sequence are important. That is, it is preferable that both sequences are selected so as to exist at a distance of 0 to 500 bases, preferably 0 to 100 bases, and most preferably 10 to 70 bases. Here, the numerical values are the F1c and F2 arrays themselves and R1
The numbers of bases not including the c sequence and the R2 sequence itself are shown.

The primer must satisfy two conditions: it can form a complementary base pair bond, and it must provide an —OH group at its 3 ′ end, which serves as a starting point for complementary strand synthesis. Therefore, the main chain is not necessarily limited to a phosphodiester bond. For example, it may be composed of a phosphothioate having a backbone of sulfur (S) instead of phosphorus (P) or a peptide nucleic acid based on a peptide bond. The base may be any one that enables complementary base pairing. In nature, it is composed of five types of adenine, guanine, cytosine, thymine and uracil, but for example, it may be an analog such as bromodeoxyuridine. The oligonucleotide used in the present invention desirably functions not only as a starting point for synthesis but also as a template for complementary strand synthesis.

The primer has a chain length that enables base pairing with a complementary strand while maintaining necessary specificity in a given environment in the following various nucleic acid synthesis reactions. . Specifically, it is 5 to 200 base pairs, more preferably 10 to 50 base pairs. Since the length of a primer recognized by a known polymerase that catalyzes a sequence-dependent nucleic acid synthesis reaction is at least about 5 bases, the length of a portion to be annealed needs to be longer. In addition, in order to expect specificity as a base sequence, it is desirable to use a length of 10 bases or more stochastically. on the other hand,
Since it is difficult to prepare an extremely long base sequence by chemical synthesis, the above-mentioned chain length is desirable. In addition, the chain length exemplified here is the chain length of the portion that anneals to the complementary strand. In the LAMP method, primers that can individually anneal to two regions (for example, F1c and F2 in the case of the FA primer), such as the above-mentioned FA primer and RA primer, are used. Therefore, the chain lengths exemplified here are the chain lengths of the respective regions constituting the primer.

Further, the primer can be labeled with a known labeling substance. As a labeling substance,
Examples include binding ligands such as digoxigenin and biotin, enzymes, fluorescent substances and luminescent substances, and radioisotopes. Alternatively, a technique of replacing a base constituting a primer with a fluorescent analog [WO95 / 05391, Pr
oc. Natl. Acad. Sci. USA, 91, 6644-6648, 1994].

The primer can be designed and synthesized so as to include an additional region. For example, an arbitrary sequence can be interposed between the F1c sequence and the F2 sequence in the FA primer. As an intervening sequence,
Examples include a restriction enzyme recognition sequence, a recognition promoter for RNA polymerase, and DNA encoding a ribozyme. By interposing a restriction enzyme recognition sequence, the obtained amplification product can be cut into double-stranded nucleic acids having the same length. In addition, by interposing an RNA polymerase recognition promoter sequence, transcription to RNA can be performed using the amplification product as a template. Furthermore, a system that cleaves a transcription product by itself is realized by interposing a DNA encoding a ribozyme. Each of these additional base sequences functions when they become double-stranded. Therefore, when the single-stranded nucleic acid of the amplification product forms a loop, these sequences do not function. It functions only when the nucleotide chain elongates and anneals to a strand having a complementary base sequence without a loop.

(5) Substrates for Amplification Reaction The substrates used in the LAMP method are usually dATP (deoxyadenosine 5'-triphosphate) and dGTP (deoxyguanosine 5'-triphosphate).
Triphosphate), dTTP (thymidine 5'-triphosphate), dCTP (deoxycytidine 5'-triphosphate), UTP (deoxyuridine
Naturally occurring nucleotide substrates such as (5'-triphosphate) are used. By using these substrates, a nucleotide chain having the same structure as that of nature can be synthesized. Also,
By using a nucleotide derivative as a substrate, it is possible to synthesize a nucleotide chain having the nucleotide derivative as a unit. Examples of the nucleotide derivative include a nucleotide labeled with a radioisotope and a nucleotide derivative labeled with a binding ligand such as biotin or digoxigenin. By using these nucleotide derivatives as substrates for a nucleotide chain amplification reaction, a labeled nucleotide chain can be generated. Similarly, a fluorescent nucleotide chain can be obtained by using a fluorescent nucleotide as a substrate. The nucleotide chain obtained here can be DNA or RNA. Which one to produce is determined by the structure of the primer, the type of substrate for the polymerization, and the combination with the polymerization reagent for polymerizing the nucleic acid.

(6) Polymerase used for amplification reaction In the LAMP method, it is essential to use a polymerase that catalyzes a strand displacement type complementary strand synthesis reaction (also referred to as a strand displacement type polymerase). Such polymerases include Bst DNA polymerase, Bca (exo-) DNA polymerase, Klenow fragment of DNA polymerase I, Vent
DNA polymerase, Vent (Exo-) DNA polymerase (Vent
DNA polymerase without exonuclease activity), DeepVent DNA polymerase, DeepVent (Exo-) DN
A polymerase (excluding exonuclease activity from DeepVent DNA polymerase), Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA
Polymerase (Takara Shuzo), KOD DNA polymerase (Toyobo) and the like. Various mutants of these enzymes can also be used in the present invention as long as the mutants have a sequence-dependent complementary strand synthesis activity and a strand displacement activity. As used herein, the term “mutant” refers to a product obtained by extracting only the portion responsible for catalytic activity,
It refers to those whose catalytic activity, stability, or heat resistance is modified by substitution, addition, or the like. However, the present invention is not limited to these.

The nucleotide chain amplification reaction in the present invention is different from the conventional PCR method in that high temperature (for example, 90 to 95 ° C.)
Does not require a double-strand dissociation step, and allows the amplification reaction to be performed at a relatively low temperature (for example, 20 to 80 ° C.) at a constant temperature. Therefore, unlike the PCR method, the DNA polymerase does not necessarily need to be a thermostable enzyme. However, the amplification reaction may be performed at a temperature of 60 ° C. or higher due to adjustment of the melting temperature (Tm). Furthermore, when double-stranded DNA is used as a template for the amplification reaction, heat denaturation may be performed to provide the first template single-stranded nucleic acid. In such cases, heat-resistant chain-substituted
Preferably, a DNA polymerase is used. Examples of the heat-resistant strand displacement DNA polymerase include Bst DNA polymerase, Bca (exo-) DNA polymerase, and Vent (Exo-) DNA polymerase.

Meanwhile, a complementary strand synthesis reaction involving strand displacement by DNA polymerase is a single-stranded binding protein (singl
e strand binding protein) is known to be promoted [Paul M. Lizardi et al, Nature Ge
netics 19, 225-232, July, 1998]. Therefore, at the time of the nucleotide chain amplification reaction, a single-stranded binding protein may be added to the reaction system for the purpose of promoting the complementary strand synthesis reaction. For example, for a complementary strand synthesis reaction by Vent (Exo-) DNA polymerase, T4 g is used as a single-stranded binding protein.
ene 32 is effective.

Furthermore, when a DNA polymerase having no 3′-5 ′ exonuclease activity is used, complementary strand synthesis does not stop at the 5 ′ end of the template and is 1% lower than the template.
It is known that a complementary strand with many bases can be obtained [C
lark JM, Joyce CM and Beardsley GP: J. Mol. Biol. 1
98 (1), 123-127, 1987]. In the present invention, such a phenomenon is undesirable because the sequence at the 3 ′ end when the complementary strand synthesis reaches the end leads to the start of the next complementary strand synthesis. However, the addition of a base to the 3 'end by DNA polymerase is A with a high probability. Therefore, the sequence may be selected so that the synthesis from the 3 'end starts at A so that there is no problem even if one base is added by mistake to dATP. In addition, even if the 3 'end protrudes during the synthesis of the complementary strand, the 3' → 5 'exonuclease activity can be utilized by digesting the 3' end and making it a blunt end. For example, natural type
Vent DNA polymerase has this activity,
This problem can be avoided by using the mixture with Vent (Exo-) DNA polymerase.

(7) Detection of Difference between Gene Sequences by LAMP Method By using the LAMP method, a difference in base sequence at a specific site between mutually homologous polynucleotide chains can be detected accurately and with high sensitivity. . That is, when detecting whether a base at a specific site on a polynucleotide chain (also referred to as a “difference site to be detected”) is the same between a first polynucleotide chain and a second polynucleotide chain, First, in the selection of an arbitrary sequence on the nucleotide chain (the above (3)), the arbitrary sequences F1c, F2c, F3
c, R1, R2, R3, or F2c-R2 are selected to include the different sites to be detected. The first and second polynucleotide chains are contacted with a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the first polynucleotide chain. The nucleotide chain analog binds tightly because it is completely complementary to the first polynucleotide chain. On the other hand, when the base at the different site to be detected in the second polynucleotide chain is the same base as the first polynucleotide chain, the nucleotide chain analog binds tightly similarly to the first polynucleotide chain. However, when the base at the different site to be detected is different from the first polynucleotide chain, the nucleotide chain analog has a lower melting point due to mispairing at the different site to be detected, and the first strand has a lower melting point. Weaker binding than with polynucleotide chains. As a result, in the amplification of a nucleotide chain in the presence of a nucleotide chain analog at a certain concentration, the rate of generation of an amplification product differs between the case where the first polypeptide chain is used as a template and the case where the second polypeptide chain is used as a template. Differences can occur. In other words, when the concentration of the nucleotide chain analog at the time of the amplification reaction is increased in order, at a certain concentration or more, a sequence having a sequence completely complementary to the nucleotide chain analog is obtained.
When one polynucleotide chain is used as a template, amplification is blocked, and when a second polynucleotide chain having a base different from the first polynucleotide at a different site to be detected is used as a template, amplification is not performed. proceed. Therefore, as a result, by examining the presence or absence of the amplification product, it becomes possible to detect a difference in the base sequence between the first nucleotide chain and the second polynucleotide chain.

More specifically, as shown in FIG. 7, the arbitrary sequence F1c is selected so as to include the different site A to be detected in the first polynucleotide chain. Then, this F1
A nucleotide chain analog (for example, a peptide nucleic acid) containing a sequence complementary to c is added to a reaction system containing the first polynucleotide chain and the second polynucleotide chain as templates. Since the nucleotide chain analog is completely complementary to F1c of the first polynucleotide chain,
Strongly binds to the c sequence. On the other hand, in the reaction system containing the second polynucleotide chain, if the difference site N to be detected on the second polynucleotide chain is A as in the first polynucleotide chain, the nucleotide chain analog is F1c
Strongly binds to the sequence, but if not A, the binding affinity of the nucleotide chain analog to the F1c sequence is reduced. As a result, under a certain amplification reaction condition, as shown in FIG. 7, when N = A, the amplification reaction is inhibited and blocked by the strong binding of the nucleotide chain analog to the F1c sequence. If N ≠ A, the binding strength of the nucleotide chain analog to the F1c sequence is not as high as N = A, and the amplification reaction proceeds. Therefore, by examining the presence or absence of the generation of an amplification product, it is possible to determine whether or not the different sites to be detected on the first and second polynucleotide chains are the same base. That is, in the presence of a nucleotide chain analog having a base sequence complementary to the sequence containing the base at the different site to be detected,
Performing the following nucleotide chain amplification reaction using the first polynucleotide chain and the second polynucleotide chain as templates, respectively, and using any of the first polynucleotide chain and the second polynucleotide chain as a template, an amplification product Is not obtained, the difference site to be detected on the two polynucleotide chains can be determined to be the same base, and an amplification product is obtained when the first polynucleotide chain is used as a template. When an amplification product is obtained when the second polynucleotide chain is used as a template, it can be determined that the different sites to be detected on the two polynucleotide chains are different bases.

[0038] Examples of the nucleotide chain analog include peptide nucleic acid (PNA), polythioamide, polysulfinamide, polysulfonamide and the like. In particular, PNA can be suitably used in the present invention because it has the following advantages. That is, (1) PNA binds more tightly to complementary strand DNA than DNA, so PNA / DNA duplex has higher thermal stability than the corresponding DNA / DNA duplex. For example, the melting point of a 15 base long PNA / DNA duplex is about 15 ° C. higher than the corresponding DNA / DNA duplex. (2) PNA has higher specificity for complementary DNA than DNA. For example, the decrease in melting point caused by a single base pair mismatch in a 15 base long duplex is due to DNA / DN
In the case of A duplex, the temperature is about 11 ° C, whereas in the case of PNA / DNA duplex, the temperature is about 15 ° C. (3) Since PNA has a higher solubility in an aqueous solution than DNA, it can be used at a higher concentration than DNA during the reaction. (4) Unlike DNA, PNA is not recognized by DNA polymerase, so that the nucleotide chain does not extend beyond PNA by the action of DNA polymerase. (5) DNA has acidic properties as a substance, whereas PNA is neutral. (6) PNA is not degraded by proteases and nucleases and
Has pH stability.

[0039] PNA has a structure in which the deoxyribose main chain of an oligonucleotide is substituted with a peptide main chain. Peptide backbones include N- (2-aminoethyl) glycine repeat units linked by amide bonds. FIG. 8 shows the structure of a PNA having a repeating unit of N- (2-aminoethyl) glycine as a main chain. Examples of the base to be bound to the peptide main chain of PNA include thymine, cytosine, adenine, guanine, inosine, uracil, 5-methylcytosine, thiouracil and 2,6-diaminopurine, and other naturally occurring bases, bromothymine, azaadenine and azaguanine. And the like. In addition, the length of the peptide nucleic acid used in the detection method of the present invention is usually 8 to 30 bases, preferably 10 to 20 bases.

The above-mentioned peptide nucleic acid is first produced by a known production method [WO96 / 40685], after producing a monomer to which each nucleobase is bound, and then reacting the obtained monomer according to a known general peptide synthesis method. Can be obtained. For example, the reaction may be carried out so as to have a desired base sequence using an automatic synthesizer according to a solid phase synthesis method. In the above method, commercially available reagents and raw materials can be used. If necessary, commercially available reagents can be appropriately derivatized according to a conventional method, for example, N-terminal and C-terminal which do not participate in the reaction of the amino acid used as the raw material. , A side chain functional group or the like for protection. The obtained nucleobase-bound PNA can be easily isolated and purified by ordinary separation means such as high performance liquid chromatography, electrophoresis chromatography, solvent extraction, and salting out.

In the amplification reaction, a buffer for giving a pH suitable for the enzymatic reaction, salts necessary for maintaining or annealing the catalytic activity of the enzyme, a protective agent for the enzyme, and, if necessary, a melting temperature (Tm) It is preferably carried out in the co-presence of a regulator or the like. As the buffering agent, one having a neutral to weak alkaline buffering action such as Tris-HCl is used. The pH is adjusted according to the DNA polymerase used. As salts, KCl, NaCl, (NH4) 2SO4 and the like are appropriately added for maintaining the activity of the enzyme and adjusting the melting temperature (Tm) of the nucleic acid. Bovine serum albumin and saccharides are used as enzyme protecting agents. Further, as a modifier for the melting temperature (Tm), dimethyl sulfoxide (D
MSO), formamide, betaine (N, N, N, -trimethylglyci
ne) and tetraalkylammonium salts and the like can be used. By utilizing a melting temperature (Tm) adjuster, the annealing of the oligonucleotide can be adjusted under limited temperature conditions. In particular, betaine
(N, N, N, -trimethylglycine) and tetraalkylammonium salts are also effective in improving the strand displacement efficiency by their isostabilize action. Betaine is 0.2-3.0 in the reaction solution.
M, preferably about 0.5 to 1.5 M, can be expected to promote the nucleic acid amplification reaction of the present invention. Since these melting temperature modifiers act in the direction of lowering the melting temperature,
Considering other reaction conditions such as salt concentration and reaction temperature, conditions for providing appropriate stringency and reactivity are empirically set.

An important feature of the LAMP method is that a series of reactions does not proceed unless the positional relationship among a plurality of regions is always maintained. By this feature, non-specific synthesis reaction accompanying non-specific complementary strand synthesis can be effectively prevented. That is, even if any non-specific reaction occurs, the possibility that the product becomes a starting material for the subsequent amplification step is reduced. The fact that the progress of the reaction is controlled by more regions leads to the freedom to construct a detection system that enables strict identification of similar base sequences.

Although the amplified nucleotide chain is composed of a complementary base sequence, although it is a single strand, most of the base chain forms a base pair bond. Utilizing this feature, amplification products can be detected. Ethidium bromide, SY
When the LAMP method is performed in the presence of a fluorescent dye that is a double-strand-specific intercalator such as BR Green I or Pico Green, an increase in the fluorescence intensity is observed with an increase in the product. By monitoring this, it is possible to track the amplification reaction in a closed system in real time. This type of detection system is PCR
Although application to the method has been considered, it is said that there are many problems since it is indistinguishable from the generation of a signal by a primer dimer or the like. However, when applied to the present invention,
Since the possibility of increasing nonspecific base pairing is very low, high sensitivity and low noise can be expected at the same time.
As with the duplex-specific intercalator, fluorescence energy transfer can be used as a method for realizing a homogeneous detection system.

According to a preferred embodiment of the LAMP method, a large number of base-pairable loops are provided on one nucleotide chain. This means that a large number of probes can hybridize to one molecule of nucleic acid, and enables highly sensitive detection. In addition, the present invention enables a nucleic acid detection method based on not only sensitivity but also a special reaction principle such as an agglutination reaction. For example, when a probe immobilized on fine particles such as polystyrene latex is added to the reaction product of the present invention, aggregation of latex particles is observed with hybridization with the probe.
If the intensity of aggregation is optically measured, highly sensitive and quantitative observation is possible. Alternatively, since the agglutination reaction can be visually observed, a reaction system that does not use an optical measurement device can be configured.

(8) Application of the method for detecting differences between gene sequences based on the LAMP method The detection method of the present invention can be applied to the detection of point mutations and single nucleotide polymorphisms on nucleotide chains. That is, many genetic diseases are known to be caused by point mutation. In order to prevent, test and diagnose these genetic diseases, it is important to investigate whether or not point mutations exist in the causative gene. The detection method of the present invention can be suitably used for detecting such a point mutation. In addition, there are patients who show side effects and patients who do not show side effects even if the same medicine is administered. A major cause of this individual difference is attributed to single nucleotide polymorphism. Therefore, by identifying the genotype of a single nucleotide polymorphism site in each individual, so-called tener-made medicine (also referred to as custom-made medicine), in which the optimal drug is administered to each patient in the optimal amount and at the correct time, is possible. Becomes The detection method of the present invention can be suitably used for detecting such a single nucleotide polymorphism.

2. Kit for detecting a difference between gene sequences of the present invention In the method for detecting a difference between gene sequences of the present invention, reagents necessary for carrying out the detection method can be packaged and supplied as a kit. More specifically, for example, in a first polynucleotide strand (control nucleotide strand) and a second polynucleotide strand (nucleotide strand to be tested) which are homologous to each other, a specific base site on the second polynucleotide strand is In the case of a kit for detecting whether or not the base is different from the base at the corresponding site on the first polynucleotide chain, a kit containing the following components may be mentioned.

[Components of Kit] (a) Nucleotide chain analog (eg, peptide nucleic acid) having a base sequence complementary to a sequence containing a base at a different site to be detected in the first polynucleotide chain (b) The first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third arbitrary sequence F3c are respectively selected from the 3 ′ end of the target region on the first polynucleotide chain toward the 3 ′ end of the polynucleotide chain. The fourth arbitrary sequence R1, the fifth arbitrary sequence R2, and the sixth arbitrary sequence R1 are sequentially arranged from the 5 'end of the target amplification region toward the 5' end of the polynucleotide chain.
When an arbitrary sequence R3 is selected, a primer containing the sequence F2 complementary to the F2c and a sequence identical to the F1c on the 5 ′ side of the F2, a primer containing the sequence F3 complementary to the F3c, A primer containing the same sequence as the R2 and a sequence R1c complementary to the R1 on the 5 ′ side of the sequence, and a primer containing the same sequence as the R3. (C) a strand displacement type complementary strand synthesis reaction A DNA polymerase that catalyzes, and (d) a nucleotide that serves as a substrate for element (c).

The components of the kit can vary depending on the embodiment of the LAMP method used. For example, in the case of the LAMP method of the embodiment of FIGS. 5 and 6, a primer containing the same sequence as R2 of the above component (b) and a sequence R1c complementary to the above R1 on the 5 ′ side of the above sequence is used. Instead, a primer containing the same sequence as R1 is used. In addition, the kit may further contain, if necessary, a buffer for giving conditions suitable for the enzymatic reaction and reagents necessary for detecting a synthesis reaction product.

Further, in a preferred embodiment of the present invention, since it is not necessary to add the reagent during the reaction, the reagent necessary for one reaction is supplied in a state of being dispensed to the reaction vessel, whereby the sample is added. The reaction can be started only by the reaction. Furthermore, by using a system in which a reaction product can be detected in a reaction vessel using a luminescence signal or a fluorescence signal, the opening of the vessel after the reaction can be completely abolished. This is very desirable in preventing contamination.

[0050]

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples, but it is apparent that the present invention is not limited to the following examples. [Example 1] Amplification of a region in M13mp18 Using M13mp18 (SEQ ID NO: 1) as a template, amplification of a partial region in M13mp18 was attempted. M13FA primer, M13RA primer, M13F3 primer and M13R3 used in the amplification reaction
The characteristics of the primers are shown in Table 1, and the positional relationship of the primers to the target sequence is shown in FIG. M13FA primer and M
The 13RA primer is an inner primer, and the M13F3 and M13R3 primers are outer primers for performing strand displacement DNA synthesis on a nucleotide chain obtained using the M13FA primer and the M13RA primer as synthesis starting points, respectively. Since the outer primer is a primer to be used as a starting point for complementary strand synthesis after the M13FA primer (or M13RA primer), anneal to the region adjacent to the M13FA primer (or M13RA primer) using the continuous stacking phenomenon. Designed to. The concentrations of these primers were set high so that annealing of the M13FA primer (or M13RA primer) occurred preferentially.

[0051]

[Table 1]

With such a primer, M13mp18
A nucleic acid is synthesized in which a region from region F1c to region R1c and its complementary base sequence are alternately linked on a single strand with a loop-forming sequence containing F2c interposed therebetween. The composition of a reaction solution for the method for synthesizing a nucleic acid according to the present invention using these primers is shown below.

(Reaction solution composition) 20 mM Tris-HCl pH8.8 10 mM KCl 10 mM (NH4) 2SO4 6 mM MgSO4 0.1% Triton X-100 5% dimethyl sulfoxide (DMSO) 0.4 mM dNTP Template DNA: 13mp18 dsDNA (SEQ ID NO: 1) Primers: 800 nM M13FA (SEQ ID NO: 2) 800 nM M13RA (SEQ ID NO: 3) 200 nM M13F3 (SEQ ID NO: 4) 200 nM M13R3 (SEQ ID NO: 5) Reaction volume 25 μl

The above reaction solution was heated at 95 ° C. for 5 minutes,
A was denatured to a single strand. Transfer the reaction solution to ice water,
4U of st DNA polymerase (NEW ENGLAND BioLabs)
The mixture was added and reacted at 65 ° C. for 1 hour. After the reaction, the reaction was stopped by maintaining the temperature at 80 ° C. for 10 minutes, and then transferred to ice water again.

Next, 1 μL of loa was added to 5 μL of the above reaction solution.
Add ding buffer and add 2% agarose gel (0.5% TB
After electrophoresis at 80 V for 1 hour using E), the gel is
Nucleic acid was confirmed by staining with reenI (Molecular Probes, Inc.). As a molecular size marker, XIV (100bp la
dder, Boehringer Mannheim). Figure
10 Lane 1 is XIV size marker, Lane 2 is 1f
mol M13mp18 dsDNA, lane 3 is control without target. In lane 3, no band was confirmed except for the unreacted primer being stained. In the case of the target, the product was confirmed as a ladder of a low-size band, a smear of a high-size band, and a band that hardly migrated in the gel when the target was present. Among the low-size bands, the bands near 290 bp and 450 bp are those in which SEQ ID NO: 6 and SEQ ID NO: 7, which are products expected by the synthesis reaction of the present invention, are double-stranded (L in FIG. 4). And P correspond to a double strand) and SEQ ID NO: 8 (corresponding to a single O long strand in FIG. 4), and the reaction proceeds as expected. It was confirmed that. The high-sized smeared pattern and the unmigrated band indicate that the reaction product is not of a certain size because the reaction is basically continuous, and that the partial single-stranded or It was thought that such an electrophoresis result was given as a result because of the complex structure in which a double-stranded complex was formed.

Example 2 Confirmation of Restriction Enzyme Digestion of Reaction Product In order to clarify the structure of the amplification product obtained in Example 1, restriction enzyme digestion was performed. If the digestion with restriction enzymes generates fragments as expected, but the high-sized smeared pattern observed in Example 1 and the bands that do not migrate disappear, these are all complementary to the single strand synthesized according to the present invention. It can be presumed that the nucleic acid is a nucleic acid in which basic nucleotide sequences are alternately linked.

Eight reaction mixtures (200 μL) were pooled, treated with phenol, and purified by ethanol precipitation. The precipitate was collected, redissolved in 200 μL of TE buffer, and 10 μL of the precipitate was digested with restriction enzymes BamHI, PvuII, and HindI.
Each was digested with II at 37 ° C. for 2 hours. The digest was electrophoresed on a 2% agarose gel (0.5% TBE) for 1 hour at 80 mV. SUPER LADDER-LOW as a molecular size marker
(100 bp ladder) (manufactured by Gensura Laboratories, Inc.) was used. Run the gel after electrophoresis with SYBR Green I (Molecular Prob
es, Inc.) to confirm the nucleic acid. The results are shown in FIG. Lane 1 is XIV size marker, Lane 2 is purified B
AmHI digest, lane 3 is a PvuII digest of the purified product, and lane 4 is a HindIII digest of the purified product.

Here, the nucleotide sequences constituting the amplification product having a relatively short chain length are estimated to be SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, etc. Table 1 shows the size of each restriction enzyme digest fragment estimated from these nucleotide sequences. L in the table is a loop (single strand)
Indicates that the migration position is undetermined.

[0059]

[Table 2]

Most of the undigested band changed to a band of an estimated size after digestion, confirming that the desired reaction product was amplified. It was also shown that there were few non-specific products.

Example 3 Promotion of Amplification Reaction by Adding Betaine Betaine (betaine: N, N, N, -trimethyl)
An experiment was conducted to examine the effect of the addition of glycine and SIGMA) on the amplification reaction of nucleic acids. As in Example 1, M13mp1
The method for synthesizing a nucleic acid in which relative base sequences are alternately linked on a single strand using the template No. 8 as a template was carried out in the presence of various concentrations of betaine. The primers used in the experiment are the same as those used in Example 1. The amount of template DNA was 10 ^-21 mol (M13mp18), and water was used as a negative control. The betaine to be added was added to the reaction solution so that the concentrations became 0, 0.5, 1, and 2 M. The composition of the reaction solution is shown below.

(Reaction solution composition) 20 mM Tris-HCl pH8.8 4 mM MgSO4 0.4 mM dNTPs 10 mM KCl 10 mM (NH4) 2SO4 0.1% TritonX-100 Template DNA: M13mp18 dsDNA (SEQ ID NO: 1) Primer: 800 nM M13FA (SEQ ID NO: 2) ) 800 nM M13RA (SEQ ID NO: 3) 200 nM M13F3 (SEQ ID NO: 4) 200 nM M13R3 (SEQ ID NO: 5) Reaction volume 25 μl

The polymerase used, reaction conditions, and electrophoresis conditions after the reaction are the same as those described in Example 1. The results are shown in FIG. In the reaction in the presence of betaine concentrations of 0.5 and 1.0 M, the amount of amplification product increased. On the other hand, when the concentration was increased to 2.0 M, no amplification product was confirmed. This showed that the amplification reaction was promoted in the presence of an appropriate amount of betaine. The decrease in amplification product at a betaine concentration of 2 M was considered to be due to the Tm being too low.

[Example 4] Detection of point mutation on K-ras gene (1) Preparation of K-ras mutant2 (mutant type) K-ras (wild type) and K-ras
K-ras mu in which codon 12 (GGT) of AGT is replaced by 1 base
tant2 (mutant type) was used. Mutant K-rasmutant2
Was prepared using LA PCR ^™ in vitro Mutagenesis Kit (Takara Shuzo) and introducing a single base substitution. Thereafter, the sequence was confirmed by sequencing. The sequence in the F1 region is shown below. Wild type: 5'-AGTTGGAGCTGGTGGCGTA-3 '(SEQ ID NO: 12) Mutant type: 5'-AGTTGGAGCTAGTGGCGTA-3' (SEQ ID NO: 13)

(2) Primers and amplification region Primers having the following sequences were used for amplification of nucleotide chains by the LAMP method. In addition, K-ras DN of each primer
The positional relationship on A (SEQ ID NO: 14) is shown in FIG. Primer FA: 5'-CTACGCCACCAGCTCCAACTTTTTATAAGGCCT
GCTGAAAATGACT-3 '(SEQ ID NO: 15) Primer FA: 5'-CAAGAGTGCCTTGACGATACAGCTTTACCTCTA
TTGTTGGATCATATTCG-3 '(SEQ ID NO: 16) Primer F3: 5'-TGTATTAACCTTATGTGTGACATGTTC-3'
(SEQ ID NO: 17) Primer R3: 5'-ATCAAAGAATGGTCCTGCAC-3 '(SEQ ID NO: 18)

(3) Synthesis of Peptide Nucleic Acid (PNA) As shown in FIG. 13, a peptide nucleic acid having the following sequence corresponding to the sequence of the F1 region was synthesized by the Fmoc method using EXPEDITE 8909 (manufactured by PE Biosystem). did. PNA sequence: 5'-TACGCCACCAGCTCC-3 '(SEQ ID NO: 19)

(4) Gene Amplification (Reaction Composition) Addition Amount Final Concentration 10 × Thermostable Polymerase Buffer 2.5 μl X1 100 mM MgSO4 0.5 μl 2 mM 5 mM dNTPs 2 μl 0.4 mM 20 pmol / μl FA Primer 2 μl 1.6 μM 20 pmol / μl RA primer 2 [mu] l 1.6 [mu] M 5 pmol F3 primer 1 [mu] l 0.2 [mu] M 5 pmol R3 primer 1μl 0.2μM 4M Betaine 6.25μl 1M PNA 2μl k-ras DNA 2μl 0.5 × 10 -17 mol 1 × 10 -17 mol or 0.5 × 10 ^-19 mol 1 × 10 ^-19 mol DW 2.75 μl volume 25 μl

The above reaction solution was heated at 95 ° C. for 5 minutes,
A was denatured to a single strand. Transfer the reaction solution to ice water,
4U of st DNA polymerase (NEW ENGLAND BioLabs)
The mixture was added and reacted at 60 ° C. for 2 hours. After the reaction, the reaction was stopped by maintaining the temperature at 80 ° C. for 10 minutes, and then transferred to ice water again.

Next, 1 μL of loa was added to 5 μL of the above reaction solution.
Add ding buffer and add 2% agarose gel (0.5% TB
After electrophoresis at 80 V for 1 hour using E), the gel is
Nucleic acid was confirmed by staining with reenI (Molecular Probes, Inc.). As a molecular size marker, XIV (100bp la
dder, Boehringer Mannheim). Figure
14 The electrophoresis image on FIG. 14 is 1 × 10 ⁻¹⁷ mol / tube
Figure shows the results when the reaction was performed in the presence of the template k-ras DNA.
14 Electrophoresis image below shows 1 × 10 ^-19 mol / tube template k-ras DNA
It shows the results when the reaction was performed in the presence. In each case, lanes 1 to 8 show the wild-type k-ras gene, and lanes 9 to 1
6 shows a case where the mutant k-ras gene was used as a template. Also,
Lanes 1 and 9 have a PNA concentration of 0 pmol / tube and lanes 2 and 10 have a PNA concentration of 0 pmol / tube.
NA concentration 0.1 pmol / tube, lane 3 and 11 PNA concentration 0.4 pmol / tube
tube, lanes 4 and 12, PNA concentration 1.6 pmol / tube, lane 5
And 13: PNA concentration 6.25 pmol / tube, lanes 6 and 14: PNA concentration
Lanes 7 and 15 have a PNA concentration of 100 pmol / tu
be, lanes 8 and 16 are for a PNA concentration of 200 pmol / tube. In FIG. 14, in lanes 7 and 8, the amplification products found in lanes 1 to 6 are not found, while the same PN
In lanes 15 and 16 at the A concentration, amplification products were detected as in lanes 9 to 14. In the lower part of FIG. 14, lane 4
In ~ 8, the amplification product seen in lanes 1-3 is not seen, whereas in lanes 12-15, the amount of amplification product decreases with increasing PNA concentration, but was detected. From the above, it was found that the difference between gene sequences based on a single base difference can be detected by the method for detecting a difference between gene sequences of the present invention.

[0070]

According to the present invention, there is provided a method for accurately and sensitively detecting differences between gene sequences.

[0071]

[Sequence List] SEQUENCE LISTING <110> Eiken Kagaku Kabushiki Kaisha <120> Method for Detection of difference among genes. <130> P00-0236 <160> 19 <210> 1 <211> 600 <212> DNA <213> Bacteriophage M13mp18 <400> 1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 240 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 480 cggaaagctg gctggagtgc gatcttcctg aggccgatac ggtcgtcgtc ccctcaaact 540 ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca 600 <210> 2 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesizedprimer sequense <400> 2 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg at 52 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 3 acaacgtcgt gactgggaaa accctttcctccgc 51 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 4 actttatgct tccggctcgt a 21 <210> 5 <211> 17 <212> DNA <213 > Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 5 gttgggaagg gcgatcg 17 <210> 6 <211> 293 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 6 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcg 293 <210> 7 <211> 293 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 7 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgt 293 <210> 8 <211> 459 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 8 acaacgtcgt gactgttggg acccttttg tgcgcccgcggtcggcgcggcgcggcgcggcgggcgcccgcggcgggcgcccgcggcgggcgggcgggcgggcgggcgcgcccgcggcgggcgggcgggcgggcgggcgggcgggacg tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 ttacccaact taatcgcctt gcagcacatc ccc ctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgt 459 <210> 9 <211> 458 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 9 cgactctaga ggcat gagcca tcagg gtcag gatcag gtcag gatcag gattcgg gtcatt gattcgg gtcatt gttcag gattcgg gtcatt gagca tcag gatcag gtcga tg tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctc tagagtcg 458 <210> 10 <211> 790 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 10 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattagcccagtcag gccagcgtaccctgcc caagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag 480 cttgcatgcc tgcaggtcga ctctagagga tccccgggta ctttttgttg tgtggaattg 540 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa ttcgagctcg 600 gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt 660 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 720 ccccctttcg ccagctggcg taatagcgaa gaggcccgca caaaaagggt tttcccagtc 780 acgacgttgt 790 <210> 11 <211> 789 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized sequence <400> 11 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcct ct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg 480 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tttttgtgcg ggcctcttcg 540 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca 600 gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag 660 gtcgactcta gaggatcccc gggtaccgag ctcgaattcg taatcatggt catagctgtt 720 tcctgtgtga aattgttatc cgctcacaat tccacacaac aaaaagtacc cggggatcct 780 ctagagtcg 789 <210> 12 <211> 19 <212> DNA <213> Bacteriophage M13mp18 <400 > 12 agttggagct ggtggcgta 19 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 13 agtt ggagct agtggcgta 19 <210> 14 <211> 420 <212> DNA <213> Homo sapiens <400> 14 ctgggtgaga ggggtctgca ggtactggtg gagtatttga tagtgtatta accttatgtg 60 tgacatgttc taatatagtc acattttcat tatttttatt ataaggcctg ctgaaaatga 120 ctgaatataa acttgtggta gttggagctg gtggcgtagg caagagtgcc ttgacgatac 180 agctaattca gaatcatttt gtggacgaat atgatccaac aatagaggta aatcttgttt 240 taatatgcat attactggtg caggaccatt ctttgataca gataaaggtt tctctgacca 300 ttttcatgag tatcaccatt ttacattccc accagcaatg cacaaagatt tcagtgtctg 360 tatccttgct aacactt <tt> ttt <tt> tgagttttttt <tt> tgagttttttt <tt> tgagttttttt <400> 15 ctacgccacc agctccaact ttttataagg cctgctgaaa atgact 46 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 16 caagagtgcc ttgacgatac agctttacct ctattgttgg atcatatt 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 17 tgtattaacc ttatgtgtga catgttc 27 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequense <400> 18 atcaaagaat ggtcctgcac 20 <210> 19 <211 > 15 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized peptide nucleic acid sequense <400> 19 tacgccacca gctcc 15

[0072]

[Sequence List Free Text]

 SEQ ID NO: 2: artificially synthesized primer sequence SEQ ID NO: 3: artificially synthesized primer sequence SEQ ID NO: 4: artificially synthesized primer sequence SEQ ID NO: 5: artificially synthesized primer sequence SEQ ID NO: 6: artificially synthesized sequence SEQ ID NO: 7: artificially synthesized sequence SEQ ID NO: 8: artificially synthesized sequence SEQ ID NO: 9: artificially synthesized sequence SEQ ID NO: 10: artificially synthesized SEQ ID NO: 11: Artificially synthesized primer SEQ ID NO: 13: Artificially synthesized primer sequence SEQ ID NO: 15: Artificially synthesized primer sequence SEQ ID NO: 16: Artificially synthesized primer sequence SEQ ID NO: 17: artificially synthesized primer sequence SEQ ID NO: 18: artificially synthesized primer sequence SEQ ID NO: 19: artificially synthesized peptide nucleus Array

[Brief description of the drawings]

FIG. 1 is a diagram showing the mechanism of sickle cell disease onset due to point mutation.

FIG. 2 is a diagram showing the process of transition from colon polyp to colon cancer based on gene mutation.

FIG. 3. Part of the principle of the strand displacement type nucleotide chain amplification method
It is a schematic diagram which shows (A)-(I).

FIG. 4. Part of the principle of the strand displacement nucleotide chain amplification method
It is a schematic diagram which shows (J)-(P).

FIG. 5: Part of the principle of the strand displacement type nucleotide chain amplification method
It is a schematic diagram which shows (a)-(g).

FIG. 6: Part of the principle of the strand displacement nucleotide chain amplification method
It is a schematic diagram which shows (h)-(n).

FIG. 7 is a schematic diagram showing one embodiment of the method for detecting a difference between gene sequences of the present invention.

FIG. 8 shows the chemical structures of PNA and DNA.

FIG. 9 is a diagram showing a positional relationship between base sequences constituting primers in a target base sequence of M13mp18.

FIG. 10 is a photograph showing the results of agarose gel electrophoresis of a product obtained by the LAMP method using M13mp18 as a template.

FIG. 11 is a photograph showing a result obtained by digesting a product obtained in Example 1 with a restriction enzyme and performing agarose gel electrophoresis.

FIG. 12 is a photograph showing the results of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid of the present invention by adding betaine using M13mp18 as a template.

FIG. 13: k-rasDNA of each base sequence constituting a primer
It is a figure which shows the positional relationship above.

FIG. 14 is a photograph showing the results of agarose gel electrophoresis of a product obtained by the LAMP method using K-ras DNA as a template in the presence of PNA.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA35 BB24 BB46 DA12 DA13 FB02 FB05 4B024 AA11 CA01 CA09 CA11 CA20 HA08 HA13 4B063 QA01 QA12 QA13 QA19 QQ03 QQ05 QQ42 QQ52 QR08 QR31 QR32 QR35 QR56 QR62QS01

Claims (19)

[Claims] 1. A method for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain which are homologous to each other, comprising the following steps: (A) a first polynucleotide chain From the 3 ′ end of the upper target region toward the 3 ′ end of the polynucleotide chain, the first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third
Are selected, and the fourth arbitrary sequence R1, the fifth arbitrary sequence R2, and the sixth arbitrary sequence R3 are sequentially arranged from the 5 ′ end of the target region toward the 5 ′ end of the polynucleotide chain. A step of selecting each, (B) the F
A sequence F2 complementary to 2c and the F2
A primer containing a sequence F3c complementary to the F3c, a primer containing a sequence F3 complementary to the F3c, a sequence identical to the R2 and a sequence R complementary to the R1 on the 5 ′ side of the sequence.
(C) preparing a primer containing 1c and a primer containing the same sequence as R3;
A first polynucleotide chain and a second polynucleotide in the presence of a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the polynucleotide chain, the primer and the polymerase; The above-described detection method, comprising: performing a nucleotide chain synthesis reaction using each of the nucleotide chains as a template; and (D) detecting the obtained amplification product. 2. A method for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain which are homologous to each other, comprising the following steps: (a) a first polynucleotide chain; From the 3 ′ end of the upper target region toward the 3 ′ end of the polynucleotide chain, the first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third
Selecting each of the fourth arbitrary sequence R1 and the fifth arbitrary sequence R3 in order from the 5 ′ end of the target region toward the 5 ′ end of the polynucleotide chain, (b) A) a sequence F2 complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2, and a primer containing the same sequence as the R1 and a primer containing the same sequence as the R3, respectively. (C) preparing a first nucleotide sequence in the presence of a nucleotide chain analog having a base sequence complementary to a sequence containing a base at a different site to be detected in the first polynucleotide chain, the primer and a polymerase; Performing a nucleotide chain synthesis reaction using each of the polynucleotide chain and the second polynucleotide chain as templates, and (d) obtaining the resulting amplified product. It said detection method comprising the step of detecting. 3. The detection method according to claim 1, wherein the nucleotide chain analog is a peptide nucleic acid. 4. The detection method according to claim 1, wherein the nucleotide chain analog comprises 8 to 30 bases. 5. F1c, F2c, F3c, R1, R2, R
The detection according to any one of claims 1 to 4, wherein 3, or the region of F2c to R2 contains a difference in base sequence to be detected between the first polynucleotide chain and the second polynucleotide chain. Method. 6. The method according to claim 1, wherein the nucleotide chain synthesis reaction is carried out using a strand displacement polymerase.
The detection method according to any one of the above. 7. The nucleotide chain synthesis reaction is carried out in the presence of a melting temperature adjusting agent.
The detection method according to any one of the above. 8. The detection method according to claim 7, wherein the melting temperature adjusting agent is betaine, dimethyl sulfoxide, formamide, or a tetraalkylammonium salt. 9. The method according to claim 8, wherein betaine is at a concentration of 0.2 to 3M. 10. The detection method according to claim 1, wherein the difference in the nucleotide sequence is caused by a point mutation. 11. The detection method according to claim 1, wherein the difference in the nucleotide sequence is caused by a single nucleotide polymorphism. 12. A kit for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain which are homologous to each other using a nucleotide chain amplification reaction, comprising: Kit containing elements. (I) a nucleotide chain analog having a base sequence complementary to a base containing a base at a different site to be detected in the first polynucleotide chain; (II) a target region on the first polynucleotide chain; The first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third arbitrary sequence F3c are respectively selected from the 'end toward the 3' end of the polynucleotide chain, and the polynucleotide is selected from the 5 'end of the target amplification region. A fourth arbitrary sequence R in order toward the 5 'end of the nucleotide chain;
1. When the fifth arbitrary sequence R2 and the sixth arbitrary sequence R3 are respectively selected, the sequence F2 complementary to the F2c is selected.
And a primer containing a sequence identical to the F1c on the 5 ′ side of the F2, a primer containing a sequence F3 complementary to the F3c, a sequence identical to the R2, and complementary to the R1 on the 5 ′ side of the sequence. A primer containing a specific sequence R1c, a primer containing the same sequence as R3, (III)
A polymerase that catalyzes a strand displacement type complementary strand synthesis reaction, and (IV) a nucleotide that serves as a substrate for the element (III). 13. A kit for detecting a difference in base sequence between a first polynucleotide chain and a second polynucleotide chain, which are homologous to each other, by using a nucleotide chain amplification reaction, comprising: Kit containing elements. (I) a nucleotide chain analog having a base sequence complementary to a base including a base at a different site to be detected in the first polynucleotide chain; (ii) a target region 3 on the first polynucleotide chain The first arbitrary sequence F1c, the second arbitrary sequence F2c, and the third arbitrary sequence F3c are respectively selected from the 'end toward the 3' end of the polynucleotide chain, and the polynucleotide is selected from the 5 'end of the target amplification region. A fourth arbitrary sequence R1 in order toward the 5 'end of the nucleotide chain;
And when the fifth arbitrary sequence R3 is selected, a sequence F2 complementary to the F2c and a primer containing the same sequence as the F1c on the 5 ′ side of the F2, a sequence F3 complementary to the F3c A primer containing the same sequence as R1; a primer containing the same sequence as R3; (iii) a polymerase that catalyzes a strand displacement type complementary strand synthesis reaction; and (iv) a substrate of element (iii). Nucleotides. 14. The kit according to claim 12, wherein the nucleotide chain analog is a peptide nucleic acid. 15. The detection method according to claim 12, wherein the nucleotide chain analog comprises 8 to 30 bases. 16. F1c, F2c, F3c, R1, R2, R
The kit according to any one of claims 12 to 15, wherein the 3 or F2c to R2 region contains a difference in the nucleotide sequence to be detected between the first polynucleotide chain and the second polynucleotide chain. . 17. The kit according to claim 12, further comprising a detection agent for detecting a product of the nucleic acid synthesis reaction. 18. The method according to claim 1, wherein the detection agent is in a region between F2c and R2
18. The kit according to claim 17, which is a probe comprising the same base sequence as its complementary strand. 19. The kit according to claim 18, wherein the probe is labeled with a particle.