WO2006051990A1 - Diagnostic method using nucleic acid amplification - Google Patents

Abstract

A method of quickly and accurately detecting a disease-associated cell or a cell originating therefrom, which is contained in a first specimen, from a second specimen. Namely, a method of detecting a disease-associated cell or a cell originating therefrom, which is contained in a first specimen obtained from a subject, from a second specimen comprising: (a) the step of detecting a gene mutation associating the disease as described above in a genome contained in the first specimen; (b) the step of detecting the above-described gene mutation in a genome contained in the second specimen; and (c) the step of comparing the gene mutation detected in the first specimen with the gene mutation detected in the second specimen. In this method, it is pointed out that a disease-associated cell or a cell originating therefrom, which is contained in the first specimen, is detected from the second specimen in the case where the gene mutations thus detected are the same as each other.

Description

 Specification

 Diagnostic method using nucleic acid amplification

 Reference to related applications

 [0001] This patent application consists of previously filed US Provisional Application No. 60Z627,181 (filing date: November 15, 2004) and Japanese Patent Application No. 2005-129830 (filing date: 200). This is accompanied by a priority claim based on April 27, 5). The entire disclosure of these earlier patent applications is hereby incorporated by reference.

 Background of the Invention

[0002] Field of the Invention

 The present invention relates to a rapid genetic diagnosis method for cells or tissues associated with a disease.

[0003] Art of Putuo

 In various diseases, cells having properties specific to the disease are known. For example, cancer cells having various properties are known even in cancer of the same tissue. Furthermore, it is known that the effects of therapeutic drugs, cancer progression, prognosis, etc. differ depending on the nature of such cancer cells. Therefore, investigating the nature of cancer cells and classifying them by molecular biology techniques provides important information related to drug selection, treatment selection, and surgical procedure selection during surgery. .

 [0004] In order to diagnose the nature of a cell associated with a disease, it is conceivable to take a tissue from the lesion of the disease and examine the characteristics of the cells contained therein. For example, in order to diagnose cancer, it is conceivable to collect cancer tissue by biopsy and examine the characteristics of cells contained in the tissue. However, with conventional inspection techniques, it takes a long time to obtain inspection results. Therefore, there is a need for techniques that allow rapid examination of cells or tissues associated with disease.

 [0005] In recent years, molecular biological studies of diseases such as cancer have progressed, and many gene mutations specific to disease-related cells such as cancer cells have been found.

[0006] On the other hand, various nucleic acid amplification methods capable of amplifying a target region in a gene have been developed. As such a method, the most well-known PCR method (US Pat. No. 4,683,195) As well as RT-PCR (Trends in Biotechnology 10, ppl46-152, 1992), which uses RNA as a cage, as well as U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159. ing.

[0007] As a nucleic acid amplification method, an isothermal amplification method that does not require complicated temperature control as in the PCR method is known. For example, a strand displacement amplification method (SDA method; Japanese Patent Publication No. 7-114718). Publication), self-sustained sequence amplification method (3SR method), Q β replicase method (Japanese Patent No. 2710159 publication), NASBA method (Japanese Patent No. 2650159 publication), LAMP method (International Publication No. ΟθΖ28082 pamphlet) The methods described in the ICAN method (WO02Z16639 pamphlet), the rolling circle method, and the WO2004Z040019 pamphlet are known.

 [0008] Further, as a method for detecting a gene mutation using a nucleic acid amplification method, a mutation detection method using the above-mentioned LAMP method (Japanese Patent Laid-Open No. 2003-159100), described in WO 01/34838 pamphlet Methods are known.

 Summary of the Invention

 [0009] The present inventors detect a genetic mutation associated with a disease using the first specimen and the second specimen obtained from the subject as samples, and the first specimen and the second specimen. By comparing the detection results between the cells, it was found that cells related to the disease contained in the first sample or cells derived therefrom can be detected quickly and accurately in the second sample. The present invention is based on these findings.

 [0010] Therefore, an object of the present invention is to provide a method for rapidly and accurately detecting a disease-related cell or a cell derived therefrom contained in a first specimen in a second specimen.

The method for detecting a disease-related cell according to the present invention is a method for detecting a cell related to a disease contained in a first sample from a subject or a cell derived therefrom in a second sample. ,

 (a) detecting a genetic mutation associated with the disease in the genome contained in the first specimen;

(b) a step of detecting the gene mutation in the genome contained in the second specimen; Bini

 (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation

 A disease-related cell contained in the first specimen or a cell derived therefrom is detected by the second specimen force when the detected gene mutation is identical to each other. It is.

 [0012] According to the present invention, a plurality of specimens collected at different times, or a plurality of specimens collected from different parts of a living body are used for disease-related cells or cells contained in one specimen. It is possible to quickly and accurately examine whether or not cells derived from this are contained in other specimens. In particular, since the detection method according to the present invention can be carried out in a short time, it is possible to obtain detection results during surgery.

 Brief Description of Drawings

 [0013] [Fig. 1] Fig. 1 is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer.

 FIG. 2 is a diagram illustrating the structure of a second primer.

 FIG. 3a is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using a first primer and a second primer.

 FIG. 3b is a diagram schematically showing the action mechanism of a nucleic acid amplification reaction using the first primer and the second primer.

[4] FIG. 4 is a diagram showing mutation sites in the human _P 53 gene nucleotide sequence (SEQ ID NO: 1).

 FIG. 5 is a graph showing the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1.

 Detailed description of the invention

 [0014] In the method for detecting a disease-related cell according to the present invention, a genetic mutation associated with a disease is detected from the genome contained in each of the first specimen and the second specimen of subject power.

[0015] The first specimen may be any disease as long as it contains cells associated with the target disease. A person skilled in the art can select as appropriate. As such a first specimen, for example, a tissue obtained by collecting the force at the center of a lesion before treatment of the target disease can be used. The second sample is a sample to be examined for the presence or amount of the disease-related cells or cells derived from the first sample, and is appropriately selected by those skilled in the art according to the target disease. Is done. For example, when the target disease is cancer, the second specimen can be tissue around the primary lesion of the cancer or tissue at a site suspected of metastasis. Also, when the lesion of the disease is removed by surgical operation, the first sample may be collected by biopsy or the like before the operation, and the second sample may be collected during the operation. One sample may be collected during the operation, and the second sample may be collected by biopsy etc. after the operation.

 [0016] According to one embodiment of the present invention, the first sample is collected before the treatment of the target disease, and the second sample is collected during or after the treatment of the disease. It is supposed to be. Such treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical therapy. In this embodiment, it is possible to examine the force majeure of the disease-related cells that existed before the treatment disappeared by the treatment of the disease, or the change in the number of the cells by the treatment, thereby evaluating the therapeutic effect. Is possible.

 [0017] According to another embodiment of the present invention, the first specimen is taken from the lesion of the target disease, and the second specimen is taken from another site. It is said. In this embodiment, it is possible to examine whether or not the disease-related cells found in the lesion of the target disease are present in other sites including the periphery of the lesion, or the abundance thereof. This makes it possible to evaluate the primary focus of the disease and metastasis to other sites (for example, cancer metastasis).

[0018] A cell associated with a disease has a gene mutation associated with the disease, preferably a gene mutation specific for the disease, and is appropriately selected by those skilled in the art depending on the target disease. Gene mutations related to this are known for various diseases, and those skilled in the art can appropriately use combinations of these diseases and gene mutations. Examples of such gene mutations include mutations in the p53 gene specific for cancer. There are differences. In addition, although a cell derived from a disease-related cell is not a disease-related cell itself, it is a cell that has proliferated and proliferated in a disease-related cell at a site other than the primary lesion of the disease. Accordingly, cells derived from disease-related cells have the same genotype as disease-related cells.

 [0019] According to a preferred embodiment of the present invention, the disease-related cell is a cancer cell or a carcinoma cell. In this embodiment, cancer-specific gene mutations are detected in the genomes contained in the first specimen and the second specimen. Cancer invasion or metastasis can be evaluated by using the first specimen as the primary tumor tissue and the second specimen as the tissue from the periphery of the primary lesion. In addition, cancer metastasis to the site can be evaluated by using the first specimen as a primary cancer tissue and the second specimen as a tissue with other site strength. Furthermore, whether the first specimen is a cancer tissue of the primary lesion and the second specimen is a cancer tissue present in another site, so that the cancer tissue present in that site is metastatic cancer from the primary lesion. Can be evaluated.

[0020] Many gene mutations specific to cancer cells are known, but the most common is the mutation of the p53 gene. The p53 protein, which also expresses this gene power, is a transcription factor that controls the cell cycle, apoptosis, and DNA repair, and plays a role in preventing the accumulation of mutations (such as canceration) at the individual level. In human cancer, mutations in the p53 gene are found in more than 50% of cases, of which 80% are point mutations with amino acid substitutions. This mutation rate is much higher than other known oncogenes. The p53 protein is a DNA binding protein, and many of the mutations found in cancer are concentrated in the DNA binding domain. In addition, hot spots where mutations are conspicuously concentrated in the p53 gene have been reported (Lopez M et al., Use of cytological specimens for p53 gene alteration detection in oral squamous cell carcinoma risk patients. Clin. Oncol. 2004 Aug; 16 (5): 366—70; Soussi T et al., Reassessment of the TP5 3 mutation database in human disease by data mining with a library of TP53 missens e mutations, Hum. Mutat. 25 ( 1): 6-17, 2004; Sudhir bnvastava, et al., Biomarkers for Early Detection of Colon Cancer, Clinical Cancer Research vol. 7, 1118, 2001; Tni erry Soussi, et al., Significance of TP53 Mutation in Human Cancer: A Critical Anal ysis of Mutations at CpG Dinucleotides, Human. Mutation Vol. 21, 192, 2003). Moreover, the frequency of mutation and the position where the mutation is likely to occur vary depending on the organ. Among human cancers, those with a high frequency of mutations in the p53 gene are lung cancer, colon cancer, spleen cancer, and mutations are found in nearly 70% of these cancers. For example, mutations in the p53 gene that are common in colorectal cancer are mutations at 175, 248, and 273, followed by CG, followed by 196, 213, 245, There is a tendency for mutations to concentrate at the 282nd codon. The types of point mutations also have a clear tendency, and there are overwhelmingly many transversions from GC to AT. In the detection method according to the present invention, any one or a combination of two or more of such gene mutations can be used.

 [0021] In a surgical operation for removing a cancer tissue, an actual lesion can be directly observed, and a more accurate and detailed tissue image can be observed. The information that the doctor wants to know is, for example, the degree of infiltration, the identity of their origin when multiple lesions are observed, and the possibility of metastasis. According to the detection method of the present invention, it is possible to obtain such information during surgery. For example, in lung cancer excision surgery, the primary cancer tissue is the first specimen, and the second specimen is the washing liquid obtained by washing the thoracic cavity, resulting in the primary lesion present in the washing liquid. The presence or absence of cancer cells can be examined, which makes it possible to accurately grasp cancer transition. Accurate information about cancer metastasis is very useful in determining the procedure. In addition, the presence or absence of cancer invasion and metastasis can be examined by using the cancer tissue of the primary lesion as the first specimen and the yarn and tissue from the periphery of the primary lesion as the second specimen. Such information is useful for determining surgical procedures, selecting postoperative treatments, selecting drugs to be administered, and determining therapeutic effects.

 [0022] Detection of gene mutation can be performed by standard methods well known in the art, such as a method using PCR, a method using a microarray, an invader method, a TaqMan PCR method, a primer extension method, and the like.

[0023] According to a preferred embodiment of the present invention, detection of a gene mutation is performed by a nucleic acid amplification method. In the nucleic acid amplification method, it is possible to detect the mutation based on the presence or absence of the amplification product by using at least one kind of primer containing a nucleotide residue that is resistant to the mutation. For example, a ply containing a mutant type as a nucleotide residue that is resistant to mutation When a mer is used, the presence of the amplification product indicates the presence of the mutation. In such a method, it is also possible to quantify the existing mutant gene simply by detecting the presence or absence of the target gene mutation. It is also possible to know the abundance ratio of the wild type gene and the mutant gene by simultaneously carrying out a nucleic acid amplification reaction using a primer containing a wild type nucleotide residue that is effective for mutation.

 [0024] As a nucleic acid amplification method, any method known to those skilled in the art may be used, but preferably the target nucleic acid can be amplified by a reaction under isothermal conditions. Examples of such isothermal nucleic acid amplification methods include the LAMP method (International Publication No. 00Z28082 pamphlet), the I CAN method (International Publication No. 02Z16639 pamphlet), the SDA method (Japanese Patent Publication No. 7-114718), and the autonomous replication method. , NASBA method (Japanese Patent No. 2650159), TMA method, Qβ replicase method (Japanese Patent No. 2710159), International Publication No. 2004Z040019, etc.

 [0025] 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” is not only to maintain the set temperature accurately, but is allowed as long as the temperature changes to the extent that the substantial function of the enzyme and the primer is not impaired. Means that. 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 ring the target nucleic acid, for example, the reaction temperature is preferably set to a temperature near the melting temperature (Tm) of the primer or lower. Furthermore, it is preferable to set the stringency level in consideration of the melting temperature (Tm) of the primer. Therefore, this temperature is preferably about 20 ° C to about 75 ° C, and more preferably about 35 ° C to about 65 ° C.

[0026] According to a preferred embodiment of the present invention, the isothermal nucleic acid amplification method comprises a primer set capable of amplifying the target nucleic acid sequence, wherein the first primer force contained in the primer set comprises the target nucleic acid sequence. A sequence (Ac ′) that hybridizes to the sequence (A) at the 3 ′ end portion is included at the 3 ′ end portion, and is a sequence that is present 5 ′ from the sequence (A) in the target nucleic acid sequence ( The sequence (Β ′) that hybridizes to the complementary sequence (Be) of B) is the 5 of the sequence (Ac). The primer set comprising the 'side is used.

 [0027] According to a particularly preferred embodiment of the present invention, the nucleic acid amplification method developed by the present inventors is used as the isothermal nucleic acid amplification method. In the method, a primer set comprising at least two kinds of primers capable of amplifying a target nucleic acid sequence, wherein the first primer included in the primer set is a sequence of the 3 ′ end portion of the target nucleic acid sequence. Complementary of sequence (B) comprising 3 'terminal portion of hybridizing sequence (Ac) in (A) and existing in the target nucleic acid sequence V and 5' to sequence (A). A sequence (Β ′) that hybridizes to the sequence (Be) is included on the 5 ′ side of the sequence (Ac ′), and the second primer included in the primer set is complementary to the target nucleic acid sequence. A folded sequence (D-) containing two nucleic acid sequences hybridizing to the sequence (Cc ') at the 3' end portion and hybridizing to each other on the same strand. Dc ') on the 5' side of the sequence (Cc ') Wherein the primer set is used.

 In the present invention, “noblybize” means that the primer hybridizes to the target nucleic acid under stringent conditions and does not hybridize to nucleic acid molecules 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. Reference can be made to J. Sambrook, EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989). For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the primer to be used, the primer can be specifically hybridized to the target nucleic acid. Such a primer can be designed using commercially available primer construction software such as Primer3 (manufactured by Whitehead Institute for Biomedical Research). According to a preferred embodiment of the present invention, a primer that hybridizes to a target nucleic acid comprises a sequence of all or part of a nucleic acid molecule complementary to the target nucleic acid.

[0029] Fig. 1 schematically shows the mechanism of nucleic acid synthesis by the first primer. First, determine the target nucleic acid sequence in the nucleic acid to be a cage, and determine the sequence (A) at the 3 'end of the target nucleic acid sequence (A) and the sequence (B) that is 5' to the sequence (A). decide. The first primer comprises the sequence (Ac ') and further comprises the sequence (Β') on the 5 'side. Array (Ac ') Is hybridized to the sequence (A), and the sequence (Β ′) is hybridized to the complementary sequence (Be) of the sequence (Β). Here, the first primer may contain an intervening sequence that does not affect the reaction between the sequence (Ac ′) and the sequence (Β ′). When such a primer is annealed to a vertical nucleic acid, the sequence (Ac) in the primer is nobbreviated 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 (Β ′) present on the 5 ′ end side of the synthesized nucleic acid hybridizes to the sequence (Be) 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 vertical nucleic acid becomes a single strand, and another primer having the same sequence as the first primer hybridizes to this part (FIG. 1 (b)). After that, the strand displacement reaction causes an extension reaction from the newly hybridized first primer, and at the same time, the previously synthesized nucleic acid is separated from the vertical nucleic acid (FIG. 1 (c)).

 [0030] In the above mechanism of action, the phenomenon that the sequence (Β ') hybridizes to the sequence (Be) is typically caused by the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into a single strand, the dissociation of a partial force or a part of the other end that is relatively unstable begins. 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, if 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 portion (sequence (A) on the vertical nucleic acid) that was exposed due to the formation of the structure, and the polymerase immediately By performing an extension reaction, the previously synthesized strand is displaced and released, and at the same time, a new double-stranded nucleic acid can be generated.

[0031] 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 homologous nucleic acid after the complementary strand of the truncated nucleic acid is synthesized by extension of the primer, a stem loop structure is formed at the 5 'end of the synthesized complementary strand. The part of the sequence (A) on the vertical nucleic acid is Must be single stranded. 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, Ratio to X (X—Y) ZX is important. However, it is not necessary to make a single strand up to the part unrelated to the hybridization of the primer, which is present 5 ′ from the sequence (配 列) on the vertical nucleic acid. In addition, in order for a new primer to efficiently anneal to a vertical nucleic acid, it is necessary to efficiently perform the above-described stem-loop structure formation. In addition, in the hybridization between the array (Β ′) and the array (Be), the efficiency V, the formation of the stem loop structure, that is, the efficiency! The distance between (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 (Β ′) to hybridize efficiently to the sequence (Be), it is considered preferable that the number of bases between both sequences is small. On the other hand, in order for the sequence (Β ′) to hybridize to the sequence (Be) and the portion of the sequence (A) on the cage nucleic acid to be a single strand, the sequence (Β ′) and the sequence (Be) It is considered preferable that the number of bases between and is larger.

[0032] From the above viewpoint, the first primer according to a preferred embodiment of the present invention is the case where there is no intervening sequence between the sequence (Ac) and the sequence (Β ') constituting the primer. , (X—Υ) ΖΧ 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. In the following, it is designed to be more preferably 0.50 or less, and still more preferably 0.25 or less. Further, (Χ + Υ) is preferably 15 or more, more preferably 20 or more, more preferably 30 or more, and is preferably 50 or less, more preferably 48 or less, and even more preferably 42 or less.

[0033] When an intervening sequence (the number of bases is Y ') exists between the sequence (Ac) and the sequence (Β') constituting the primer, the first primer according to a preferred embodiment of the present invention {X— (Υ—Υ′ΜΖΧ is 1 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 preferably designed to be 0.75 or less, more preferably 0.50 or less, and even more preferably 0.25 or less, and (。 + Υ + Υ ′) is preferably 15 or more, more preferably Is 20 or more, more preferably 30 Also, it is preferably 100 or less, more preferably 75 or less, and still more preferably 50 or less.

 [0034] The first primer has a chain length such that base pairing with a target nucleic acid can be carried out while maintaining necessary specificity under given conditions. The chain length of this primer is preferably 15 to 100 nucleotides, more preferably 20 to 60 nucleotides. The lengths of the sequence (Ac ′) and the sequence (Β ′) constituting the first primer are each preferably 5 to 50 nucleotides, more preferably 7 to 30 nucleotides. If necessary, insert an intervening sequence that does not affect the reaction between sequence (Ac ') and sequence (Β').

 [0035] As described above, the second primer is added to the sequence (C) of the 3 'end portion 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) comprising two nucleic acid sequences hybridizing to each other on the same strand, comprising a sequence (Cc) to be hybridized (Cc) at the 3 ′ end. On the side. The structure of such a second primer is, for example, as shown in FIG. 2, but is not limited to the sequence and the number of nucleotides shown in FIG. The length of the sequence (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, further preferably 4 to 60 nucleotides, and further preferably 6 to 40 nucleotides. The number of nucleotides in the base pair formed by hybridization within the folded sequence is preferably 2 to 500 bp, more preferably 2 to 50 bp, more preferably 2 to 30 bp, more preferably 2 to 30 bp. Furthermore, it is preferably 3 to 20 bp. The nucleotide sequence of the folded sequence (D-Dc) may be! Or 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 ′).

[0036] A possible mechanism of action of the nucleic acid amplification reaction by the first primer and the second primer will be described with reference to Fig. 3 (Figs. 3a and 3b). In FIG. 3, in order to simplify the description, two sequences to be hybridized are referred to as complementary sequences. iS This does not limit the invention. 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 (one), and the 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 extension strand (one) is released. Next, the second primer hybridizes to the sequence (C) on the released extended strand (-) (Fig. 3 (c)), and the extension reaction of the primer occurs, and the extended strand (+) is synthesized. (Figure 3 (d)). A stem-loop structure is formed between the 3 'end of the generated extended strand (+) and the 5' end of the extended strand (one) (Fig. 3 (e)), and the extended strand (+) that is the free 3 'end The extension force (1) is released at the same time as the elongation at the end of the loop occurs (Fig. 3 (1)). By the extension reaction of the loop tip force, a hairpin-type double-stranded nucleic acid in which the extension strand () is bound to the 3 ′ side of the extension strand (+) via the sequence (A) and the sequence (Be) is generated. The first primer hybridizes to the sequence (A) and sequence (Be) (Fig. 3 (g)), and the extension chain (-) is generated by the extension reaction (Figs. 3 (h) and (0)). In addition, the folded sequence present at the 3 ′ end of the hairpin double-stranded nucleic acid provides a free 3 ′ end (FIG. 3 (h)), and an extension reaction therefrom (FIG. 3 (0 ), A single-stranded nucleic acid having a folded sequence at both ends and containing an extended strand (+) and an extended strand (one) alternately through the sequences derived from the first and second primers (FIG. 3). (j)) In this single-stranded nucleic acid, the folded sequence present at the 3 'end provides a free 3' end (starting point of complementary strand synthesis) (Fig. 3 (k)). Anti The chain length is doubled per extension reaction (Fig. 3 (1) and (m)), and the extended strand from the first primer released in Fig. 3 (- ) Provides a free 3 'end (the complementary strand synthesis origin) due to the folding sequence present at the 3' end (Fig. 3 (n)). A loop structure is formed, producing a single-stranded nucleic acid containing an extended strand (+) and an extended strand (one) alternately via a sequence derived from the primer (Figure 3 (o)). In the case of nucleic acids as well, since the complementary strand synthesis origin is sequentially provided by the loop formation at the 3 ′ end, extension reactions occur one after another. Sequences derived from the first and second primers are included between the extended strand (+) and the extended strand (-). Because, each primer extension reaction was Haiburidizu This can significantly amplify the sense and antisense strands of the target nucleic acid.

 [0037] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. In addition, as a way to include.

 In the present invention, “do not compete!” Means that the primer does not interfere with the provision of the complementary strand synthesis starting point by hybridizing to the target nucleic acid.

 [0039] 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. There is a folded sequence or loop structure at the 3 ′ end of the amplified product, and extension reactions occur one after another from the complementary strand synthesis starting point provided. The third primer can anneal to the target sequence present in the single-stranded part when such an amplification product is partially in a single-stranded state. As a result, a new complementary strand synthesis origin 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.

 [0040] The third primer is not necessarily limited to one type, but two or more types of the third primer may be used simultaneously in order to improve the speed and specificity of the nucleic acid amplification reaction. These third primers typically have different sequence powers than the first primer and the second primer, and may hybridize to a partially overlapping region as long as they do not compete with these primers. The chain length of the third primer is preferably 2 to: LOO nucleotides, more preferably 5 to 50 nucleotides, and even more preferably 7 to 30 nucleotides.

[0041] The third primer mainly has an auxiliary function for proceeding more rapidly with the nucleic acid amplification reaction by the first primer and the second primer. Accordingly, it is preferable that the third primer has a Tm lower than the Tm of the third and third ends of the first primer and the second primer, respectively. The amplification reaction of the third primer The amount added to the solution is preferably smaller than the amount added to each of the first primer and the second primer.

 [0042] In order to determine the presence or absence of a gene mutation using the primer set, the primer set is designed so that the mutation site is included in the sequence (A), the sequence (B), or the sequence (C). can do. Accordingly, it is possible to determine the presence or absence of the mutation by confirming the presence or absence of the amplification product, and it is possible to determine the amount of the gene having the mutation by quantifying the amplification product.

 [0043] According to one embodiment of the present invention, the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (A). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably included in the 5 ′ end of the sequence (A) (corresponding to the 3 ′ end in the first primer). The sequence (Ac ′) contained in the first primer is preferably a sequence complementary to the sequence (A).

 [0044] According to another embodiment of the present invention, the primer set is designed such that a nucleotide residue related to mutation is included in the sequence (C). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the amplification product is obtained because the second primer anneals to the sequence (C) in the nucleic acid amplification reaction. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. Therefore, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. The nucleotide residue involved in the mutation is preferably contained in the 5 ′ end of the sequence (C) (corresponding to the 3 ′ end in the second primer). The sequence (Cc ′) contained in the second primer is preferably a sequence complementary to the sequence (C).

[0045] According to another embodiment of the present invention, the primer set may comprise a mutation-related nucleotide. It is designed such that a residue is included in the sequence (B). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, in the nucleic acid amplification reaction, the first primer is annealed to the sequence (A) and the extension reaction is performed. Since the contained sequence (Β ') hybridizes to the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in FIG. 1 proceeds efficiently, resulting in an amplification product. On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence at the mutation site, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. The mechanism of action shown in 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The sequence (Β ') contained in the first primer is preferably the same sequence as the sequence (Β).

 [0046] The detection of the mutation in the above-described sequence (Β) will be described in more detail. In the mechanism of action shown in FIG. 1, the phenomenon that the sequence (Β ′) hybridizes to the sequence (Be) occurs due to the presence of complementary regions on the same strand. In general, when a double-stranded nucleic acid is dissociated into single strands, partial dissociation starts at its end or other relatively unstable partial force. In the double-stranded nucleic acid produced by the extension reaction with 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-and-loop structure can be formed as a metastable state. However, this stem loop structure does not exist stably, especially when there are non-complementary nucleotides between the sequence (Β ') and the sequence (Be) part that form the stem. Or the stem is not formed at all. In this case, the hybridization between the vertical sequence (A) and the sequence (Ac ') in the primer is more dominant, and the sequence (A) is not a single strand. The next first primer cannot be annealed. Therefore, it is extremely difficult to cause the continuous reaction shown in Fig. 1.

[0047] Further, in order to detect the presence or absence of sequence deletion or insertion in the nucleic acid sequence using the primer set, the site force sequence (A) or sequence (B) related to the deletion or insertion Alternatively, a set of primers can be designed to be placed between the force contained in sequence (C) or between sequence (A) and sequence (B). This makes it possible to determine the presence or absence of a sequence deletion or insertion by confirming the presence or absence of an amplification product, and the amount of a gene having the deletion or insertion by quantifying the amplification product. Can be determined.

 [0048] According to one embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is included in the sequence (A). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the first primer is annealed to the sequence (A) in the nucleic acid amplification reaction, so that an amplification product is obtained. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the first primer to anneal to the sequence (A) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced. The sequence (Ac) contained in the first primer is preferably a sequence complementary to the sequence (A).

 [0049] According to another embodiment of the present invention, the primer set is designed such that a site for deletion or insertion is included in the sequence (C). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the amplification product is obtained because the second primer anneals to the sequence (c) in the nucleic acid amplification reaction. If the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, it will be difficult for the second primer to anneal to the sequence (C) in the nucleic acid amplification reaction. No amplification product is obtained or the amount of amplification product obtained is significantly reduced. The sequence (Cc) contained in the second primer is preferably a sequence complementary to the sequence (C).

[0050] According to another embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is included in the sequence (B). In this embodiment, when the nucleic acid sample contains the target nucleic acid sequence, the nucleic acid amplification reaction includes the first primer after annealing to the sequence (A) and the extension reaction, and then the primer is contained in the primer. Since the sequence (Β ') hybridizes to the sequence (Be) on the extended strand, the stem loop structure It is formed efficiently. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Fig. 1 proceeds efficiently, resulting in an amplified product. . On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, formation of the stem-loop structure in the nucleic acid amplification reaction becomes difficult. The mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. The details are as described above for the mutation detection according to the present invention. The sequence (配 列 ′) contained in the first primer is preferably the same sequence as the sequence (Β).

[0051] According to a preferred embodiment of the present invention, the primer set is designed such that a site related to deletion or insertion is located between the sequence (Α) and the sequence (Β). In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, in the nucleic acid amplification reaction, the first primer is annealed to the sequence (Α) and the extension reaction is performed, and then the primer is added to the primer. Since the contained sequence (Β ') hybridizes to the sequence (Be) on the extended strand, a stem loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape, and the mechanism of action shown in Figure 1 proceeds efficiently, resulting in an amplified product. . On the other hand, if the nucleic acid sample contains a nucleic acid sequence that is different from the target nucleic acid sequence due to deletion Z insertion, the sequence (Β ') contained in the first primer and the sequence on the extended strand ( Therefore, it is difficult to form the stem-loop structure in the nucleic acid amplification reaction. Therefore, in this case, the mechanism of action shown in FIG. 1 is hindered and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced.

[0052] According to another embodiment of the present invention, the primer set is designed such that a site for deletion or insertion is located between the sequence (A) and the sequence (C). . In this embodiment, when the target nucleic acid sequence is contained in the nucleic acid sample, the primer is annealed to the sequence (A) in the nucleic acid amplification reaction and the extension reaction is performed. Since the sequence (Β ′) contained in is hybridized with the sequence (Be) on the extended strand, a stem-loop structure is efficiently formed. The formation of this efficient stem-loop structure allows the other first primer to anneal in a saddle shape. Since the mechanism of action shown in (1) proceeds efficiently, an amplification product is obtained. On the other hand, if the nucleic acid sample contains a nucleic acid sequence that differs from the target nucleic acid sequence due to deletion z insertion, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. . For example, when a long sequence is inserted between sequence (A) and sequence (C), a nucleic acid sequence that is different from the target nucleic acid sequence is included in the nucleic acid sample! Since (efficiency) is significantly reduced, no amplification product is obtained or the amount of amplification product obtained is significantly reduced. In addition, a nucleic acid sequence that differs from the target nucleic acid sequence is contained in the nucleic acid sample due to the deletion of the sequence between sequence (A) and sequence (C). If part or all is lost, the sequence (Β ') contained in the first primer cannot be hybridized on the extended strand, making it impossible or difficult to form a stem-loop structure. Therefore, the mechanism of action shown in FIG. 1 is hindered, and no amplification product is obtained, or the amount of amplification product obtained is significantly reduced. Furthermore, a nucleic acid sequence that differs from the target nucleic acid sequence due to the deletion of the sequence between sequence (Α) and sequence (C) is contained in the nucleic acid sample. Even if no deletion occurs, the speed (efficiency) of nucleic acid amplification is reduced, so that no amplification product is obtained or the amount of amplification product obtained is significantly reduced.

As the polymerase used in the nucleic acid amplification reaction, any one having room temperature, medium temperature, or heat resistance can be suitably used as long as it has strand displacement activity (strand displacement ability). Further, this polymerase may be either a natural body or a mutant having an artificial mutation. An example of such a polymerase is DNA polymerase. Further, it is preferable that this DNA polymerase has substantially no 5 → 3 exonuclease activity. Such DNA polymerases include thermophilic Bacillus such as Bacillus stearothermophilus (hereinafter referred to as “B. st”), Bacillus caldotenax (hereinafter referred to as “B. ca”), and the like. 5 ′ → 3 of DNA polymerase derived from genus bacteria, mutants lacking exonuclease activity, and Tarenow fragment of DNA polymerase I derived from E. coli. Examples of DNA polymerases used in nucleic acid amplification reactions include Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Ex o-) DNA polymerase, Φ 29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, Pfo DNA polymerase, Pfo turbo DNA polymerase, KOD DNA polymerase, 9 ° Nm DNA polymerase, Therminater DNA polymerase, etc. Can be mentioned.

 [0054] Examples of other reagents used in the nucleic acid amplification reaction include catalysts such as magnesium chloride, magnesium acetate, and magnesium sulfate, substrates such as dNTP mix, tris hydrochloride buffer, tricine buffer, sodium phosphate buffer, A buffer such as potassium phosphate buffer can be used. Furthermore, additives such as dimethyl sulfoxide and betaine (Ν, Ν, Ν-trimethylglycine), acidic substances and cation complexes described in WO 99/54455 pamphlets 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 nucleic acid amplification efficiency. 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, it is possible to adjust the intensity of double strand formation in the nucleic acid. A general melting temperature adjusting agent has an effect of lowering the melting temperature. By adding such a melting temperature adjusting agent, it is possible to lower the melting temperature of the duplex forming part between two nucleic acids, in other words, to reduce the strength of the duplex formation. Is possible. 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, whereby the next primer becomes hybridized to the target region after the extension reaction by the primer is completed, so that the nucleic acid amplification efficiency can be increased. The concentration of the melting temperature adjusting agent used in the present invention and the concentration thereof in the reaction solution is determined based on other reaction conditions that affect the hybridization conditions such as salt concentration, reaction temperature, and the like. Is selected appropriately. 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). ).

 [0056] Further, in the nucleic acid amplification reaction, an enzyme stabilizer can be added to the reaction solution. As a result, the enzyme in the reaction solution is stabilized, so that the nucleic acid amplification efficiency can be increased. The enzyme stabilizer used in the present invention is not particularly limited and may be any one known in the art such as glycerol, urine serum albumin, saccharides and the like.

 [0057] 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 reagents are well known in the art and may be rugged, but are not particularly limited, but are preferably saccharides, more preferably monosaccharides or oligosaccharides, more preferably It can be trehalose, sorbitol or mannitol, or a mixture of two or more of these.

 [0058] According to a preferred embodiment of the present invention, the nucleic acid amplification reaction is carried out in the presence of a mismatch recognition protein, which makes it possible to detect mutations more accurately.

[0059] It is already known that bacteria, yeast, and the like have a mechanism for repairing a mismatched base pair that is not partially matched in the double strand of DNA. . This repair is performed by a protein called “mismatch binding protein” (also referred to as “mismatch recognition protein”). MutS protein (Japanese Patent Publication No. 9 50469 9), MutM protein (Japanese Patent Laid-Open No. 2000-2000) No. 300265) and various mismatch binding proteins such as MutS protein (International Publication No. 99/06591 pamphlet) bound to GFP (Green Fluores cence Protein) have been reported. Furthermore, in recent years, Misuma Tutsi gene diagnostic method to detect mismatches utilizes a binding protein have been developed (M. Gotoh et al., Genet . Anal, 14, 47-50, 1997) specific in ₀ in a nucleic acid As a method for detecting polymorphisms and mutations in a nucleotide, for example, a mismatched control nucleic acid is hybridized with a test nucleic acid suspected of having a mutation, and a mismatch recognition protein is introduced into the mismatched nucleic acid. There are known methods for detecting.

[0060] In the present invention! / “Mismatch” means adenine (A), gyonin (G), cytosine (C), and Means that one base pair selected from thymine (T) (uracil (U) in the case of RNA) is not a normal base pair (A and T combination or G and C combination) . Mismatches include not only one mismatch but also multiple consecutive mismatches, mismatches caused by insertion and Z or deletion of one or more bases, and combinations thereof.

 [0061] The specificity (accuracy) can be improved also in a nucleic acid amplification reaction for detecting a gene mutation by using these mismatched binding proteins. In the case of a nucleic acid amplification reaction, a small amount of a heterozygous double-stranded structure may occur between two nucleotide sequences that are not complementary to each other. In the present invention, the term “heteroduplex structure” means a double-stranded structure containing a non-complementary region by having one or more mismatches, which is a substantially complementary double-stranded structure. . Such a heteroduplex structure results in a false amplification product that should not be produced by nature. Therefore, if a mismatch binding protein is added to the reaction solution used in the nucleic acid amplification reaction, the mismatch binding protein binds to the heteroduplex structure as described above, and the subsequent amplification reaction is prevented. Therefore, by using a mismatch binding protein, it is possible to prevent the generation of an erroneous amplification product.

[0062] The mismatch binding protein used in the present invention may be any protein known to those skilled in the art as long as it is a protein capable of recognizing a mismatch in a double-stranded nucleic acid and binding to the mismatch site. It may be a thing. In addition, the mismatch-binding protein used in the present invention has one or more amino acid substitutions, deletions, additions, and / or insertions in the amino acid sequence of the wild-type protein as long as the mismatch in the double-stranded nucleic acid can be recognized. It may be a protein (mutant) that also has an amino acid sequence ability! /. Such mutants can also be created artificially by forces that may occur in nature. Many methods are known for introducing amino acid mutations into proteins. For example, site-directed mutagenesis methods include WP Deng and JA Nickoloff's method (Anal. Biochem., 200, 81, 1992), Κ 丄. Makamaye and F. Eckstein's method (Nucleic Adids Res., 14, 9679). -9698, 1986), etc., and the random mutagenesis method uses a method using Escherichia coli XLl-Red strain (Stratagene) lacking the basic repair system, sodium nitrite, etc. And methods for chemically modifying bases (J.-J. Diaz et al., BioTechnique, 11, 204-211, 1 991) are known. Many such mismatch binding proteins are known, such as MutM, Mut S and their analogs (Radman, M. et al. Annu. Rev. Genet. 20: 523-538 (1986)). Radaman, M. etal., Sci. Amer., August 1988, pp40-46; Modric h, P., J. Biol. Chem. 264: 6597-6600 (1989); Lahue.RS et al, Science 245: 160-164 (198 8); Jiricny, J. et al '. Nucl. Acids Res. 16: 7843-7853 (1988); Su, SSet al., J. Biol. Chem. 263; 6829-6835 (1988) Lahue, RSet al. 'Mutat. Res. 198: 37-43 (1988); Dohet, C. et al. Mol. G en. Gent. 206: 181-184 (1987); Jones.M. Et al. 'Gentics 115: 605-610 (1987); Muts of Salmonella typhimurium (Lu, AL, Genetics 118: 593-600 (1988); HaberL.T. Et al.J.Bact eriol.l70: 197-202 (1988) Pang, PPet al. J. Bacteriol. 163: 1007-1015 (1985)); and Pribebe SD et al., J. Bacterilo. L70: 190-196 (1988)). The mismatch binding protein used in the present invention is preferably derived from MutS, MutH, MutL, or yeast, and more preferably MutS, MutH, or MutL.

 [0063] A mismatch binding protein may also bind to a single-stranded nucleic acid, and it is known that binding of such a mismatch binding protein to a single-stranded nucleic acid is inhibited by the single-stranded binding protein. ing. Therefore, when a mismatch binding protein is used in the nucleic acid amplification reaction, it is preferable to use a single-stranded binding protein in combination. Mismatch-binding proteins can also bind to double-stranded nucleic acids that do not contain mismatches. Such misbinding of mismatch-binding proteins can be activated by using a activating agent to activate the mismatch-binding proteins. It is known that it is inhibited by keeping it. Therefore, when using a mismatch binding protein in a nucleic acid amplification reaction, it is preferable to use a protein that has been activated in advance by an activator.

[0064] The single-stranded binding protein (SSB) used to inhibit the binding of the mismatch binding protein to the single-stranded nucleic acid can be any SSB known in the art. Preferred SSBs include single-stranded binding proteins from Escherichia coli, Drosophila, and Xenopus, and gene 32 protein from T4 butteriophage, and their equivalents from other species. Can be mentioned. The mismatch binding proteins used in this case are MutS, MutH, MutL, HexA, MSH1-6, Rep3, RNaseA, uracil-DNA glycosidase, T4 endonuclease VII, resolvase, etc., preferably MutS, MSH2 or MSH6, or a mixture of two or more of these, more preferably MutS Is done.

[0065] The active agent for activating the mismatch binding protein can be appropriately selected by those skilled in the art, and is not particularly limited, but preferably ATP (adenosine 5'-triphosphate). , ADP (adenosine 5'-diphosphate), ATP-γ 3 (adenosine 5 '0 (3-thio triphosphate)), AMP-PNP (adenosine 5' [ _J 8, γ imido] triphosphate), etc. Or one of the nucleotides that can bind to the mismatch binding protein. The activity of the mismatch binding protein can be performed by incubating the mismatch binding protein and the active agent at room temperature for several seconds to several minutes.

[0066] The presence of the amplification product obtained by the nucleic acid amplification method can be detected by many various methods. One method is the detection of amplification products of a specific size by general gel electrophoresis. In this method, for example, it can be detected by a fluorescent substance such as ethidium bromide or cyber green. As another method, detection can be performed by using a labeled probe having a label such as piotin and hybridizing it to the amplification product. Piotin can be detected by binding to fluorescently labeled avidin, avidin bound to an enzyme such as peroxidase, and the like. Yet another method is to use immunochromatography. In this method, it is devised to use a chromatographic medium using a label detectable with the naked eye (Immunochromatography method). If the amplified fragment and the labeled probe are hybridized and a further different sequence of the amplified fragment and a hybridizable capture probe are fixed to the chromatographic medium, the trapped portion can be trapped. Detection with is possible. As a result, simple detection with the naked eye is possible. Furthermore, in the nucleic acid amplification method by the present inventors, the amplification efficiency in the nucleic acid amplification reaction is very high, so that the amplification product can also be detected indirectly by using the fact that pyrophosphate is generated as an amplification byproduct. As such a method, for example, there is a method of visually observing the white turbidity of the reaction solution by utilizing the fact that pyrophosphoric acid binds to magnesium in the reaction solution to cause white precipitation of magnesium pyrophosphate. The Another method is that pyrophosphate binds strongly to metal ions such as magnesium. There is a method that utilizes the fact that the magnesium ion concentration in the reaction solution is significantly reduced by forming an insoluble salt. In this method, a metal indicator whose color tone changes according to the magnesium ion concentration (for example, Eriochrome Black T, Hydroxy Naphthol Blue, etc.) is added to the reaction solution, and the color change of the reaction solution is visually observed. This makes it possible to detect the presence or absence of amplification. Furthermore, by using Calcein, etc., the increase in fluorescence accompanying the amplification reaction can be observed visually, so that amplification products can be detected in real time.

 [0067] In the detection method according to the present invention, the gene mutation detected from the first specimen is then compared with the gene mutation detected from the second specimen. In this comparison, if these detected gene mutations are identical to each other, it indicates that a disease-related cell contained in the first sample or a cell derived therefrom was detected in the second sample. It is.

 [0068] According to the detection method of the present invention, the sample collected before the treatment of the target disease is the first sample, and the sample collected during or after the treatment of the disease is the second sample. This makes it possible to evaluate the therapeutic effect. Therefore, according to the present invention, there is provided a method for evaluating the effect of treatment of a disease in a subject, the method comprising: (a) a genome contained in a first sample collected from a subject before the treatment of the disease. And (b) detecting a gene mutation in a genome contained in a second specimen collected from the subject during or after the treatment of the disease. And (c) comparing a first gene force detected gene mutation and a second sample force detected gene mutation. In this method, when the detected gene mutation is different from each other or when the gene mutation is not detected in the second specimen, it is evaluated that the therapeutic effect is high. The treatment is appropriately selected by those skilled in the art depending on the target disease, and examples thereof include drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical treatment.

[0069] Further, according to the detection method of the present invention, the sample collected from the lesion of the target disease is used as the first sample, and the sample collected from other sites is used as the second sample. It becomes possible to evaluate the primary focus of the disease and metastasis to other sites (for example, cancer metastasis). Therefore, according to the present invention, the primary lesion force of the disease in the subject is transferred to other sites. A method of evaluating, comprising: (a) detecting a genetic mutation associated with the disease in a genome contained in a first specimen collected from the primary lesion of the disease of the subject, (b) the subject A step of detecting the genetic mutation in the genome contained in the second specimen from which the other site forces were also collected, and (c) the genetic mutation detected from the first specimen and the second specimen force. Comparing the mutation. In this method, if the detected gene mutations are identical to each other, it is evaluated that there is metastasis from the primary lesion site to another site. According to a preferred embodiment of the present invention, the disease is cancer or tumor, and metastasis to these other sites is evaluated. Further, cancer or tumor infiltration can be evaluated by using a cancer tissue or tumor tissue in the primary lesion as the first specimen and a tissue in the periphery of the primary lesion as the second specimen. Further, by setting the cancer tissue of the primary lesion as the first specimen and the cancer tissue present in the other part as the second specimen, the cancer tissue present in that part is metastatic cancer from the primary lesion. It can be evaluated whether or not there is.

 Example

 [0070] Hereinafter, the present invention will be specifically described by way of examples. However, the scope of the present invention is not limited to these examples.

[0071] Example ι _: Ό Primary lesion of colorectal cancer indexed by 53 gene differences

 In this example, using the tissue obtained from colon cancer patients as a specimen, mutations in the human p53 gene (SEQ ID NO: 1) were examined by nucleic acid amplification. The mutations to be examined were nucleotide substitutions at the 175th, 213rd, 245th, 248th, and 273rd codons (boxed in Fig. 4).

 [0072] Primers having the following sequences were used in the nucleic acid amplification reaction.

 [0073] Forward primer:

175F-W: 5'-GGCAGCGCCTTCTACAAGCAGTCACAGCAC-3 '(SEQ ID NO: 2); 175F-M: 5 -GGCAGTGCCTTCTACAAGCAGTCACAGCAC-3' (SEQ ID NO: 3); 213F-W: 5 '-ATGTCGAAAAAATTTGCGTGTGGAGTATTT-3' (SEQ ID NO: 4); 213F-M: 5 -ATGTCAAAAAAATTTGCGTGTGGAGTATTT-3 '(SEQ ID NO: 5); 245F-W: 5 GATGCCGCCCATCCACTACAACTACATGTG-3 '(SEQ ID NO: 6); 245F-M1: 5 -GATGTCGCCCATCCACTACAACTACATGTG-3' (SEQ ID NO: 7); 245F-M2: 5 -GATGCTGCCCATCCACTACAACTACATGTG-3 '(SEQ ID NO: 8); W: 5 -CCTCCGGTTCAACTACATGTGTAACAGTTC-3 '(SEQ ID NO: 9); 248F-M1: 5 -CCTCTGGTTCAACTACATGTGTAACAGTTC-3' (SEQ ID NO: 10); 248F-M2: 5 '-CCTCCAGTTCAACTACATGTGTAACAGTTC-3' (SEQ ID NO: 11); 273F-W : 5 -AAACACGCACTGGTAATCTACTGGGACGGA-3 '(SEQ ID NO: 12); 273F-M1: 5 -AAACATGCACTGGTAATCTACTGGGACGGA-3' (SEQ ID NO: 13); 273F-M2: 5 '-AAAC ACGC ACTGGTAATCTACTGGGACGGA-3' (SEQ ID NO: 14).

[0074] Reverse primer:

 R1: 5'—GGATATATATATATCCAGATTCTCTTCCTCTGTGCG—3 ′ (SEQ ID NO: 15); R2: 5′—GGATATATATATATCCCTGGAGTCTTCCAGTGTGAT—3 ′ (SEQ ID NO: 16); R3: 5 GGATATATATATATCCCTCAGGCGGCTCATAGGGCA-3 ′ (SEQ ID NO: 17)

R4: 5'— GGATATATATATATCCCATCGCTATCTGAGCAGCGC— 3 '(SEQ ID NO: 18)

[0075] The number attached to the beginning of the name of the forward primer indicates the codon number in the p53 gene to be examined. The underlined portion of the forward primer sequence is a nucleotide residue that can be used for the mutation to be examined. Furthermore, “F” indicates a forward primer, and “R” indicates a reverse primer. “W” means that the primer contains a wild type sequence, and “M” means that the primer contains a mutant type sequence.

[0076] In the forward primer, the sequence at the 3, terminal side (about 20 mer) is ringed in a cage shape, and the sequence at the 5 'end (lOmer) is on the extension strand of the primer. It is designed to hybridize to the region starting 16 bases downstream of the 3 'terminal residue. The reverse primer is designed to have a structure in which the sequence at the 3rd end (20mer) anneals in a cage shape and the sequence at the 5 'end (16mer) folds within that region. . The forward primer contains nucleotide residues that are resistant to mutation Therefore, it is possible to determine whether or not a residue that can be mutated is included in the forward primer depending on the presence or absence of an amplification product by the nucleic acid amplification reaction.

 [0077] Two patients with colorectal cancer (Case 1 and Case 2) Using a biopsy needle from the central lesion, close part, remote part, metastasis, etc. of a human colon cancer tissue removed by surgery Cancer tissues were collected, and each tissue was suspended in 1 ml of water and heated at 98 ° C. for 5 minutes. A 1 μl suspension of each sample was used for the nucleic acid amplification reaction.

 [0078] The above-described tissue suspension (1 μ 1) was added to a reaction solution (24 L) having the following composition: Tris—HCl (20 mM, pH 8.8), KCl (lOmM) ゝ (NH) SO ( lOmM) ゝ MgSO (8mM), DMSO

 4 2 4 4

 (3%), Triton X—100 (1%), dNTP (1.4 mM), MutS (1 μg), 2000 nM primer pair, Cyber Green (0.01 μ1 / ml), and 8 U Contains Bst DNA polymerase (NEW ENGLAND BioLabs); and incubated at 60 ° C for 26 minutes. This amplification reaction was carried out using a real-time PCR apparatus (Stratagene).

 [0079] The graph shown in FIG. 5 shows the time course of the amplification reaction in the tissue at the center of the primary lesion in case 1. Tables 1 and 2 below summarize the results of amplification reactions for Case 1 and Case 2, respectively. In these tables, the amplification reaction results are +++, ++, +, and king, starting from the one with the highest increase rate of the amplification product (the one with the fastest rise of the curve in the graph). What did not exist was taken as one.

[0080] [Table 1]

Table 1: Results of amplification reaction for case 1

 a Woven Ford Reverse Amplification Result Primer Primer

 Central focus 175F-W R4 + + +

 175F-M R4 ―

 213F- R3 + + +

213F-M R3 ―

 245F-W R2 + + +

245F-M1 R2 ―

 245F-M2 R2 ―

 248F- R2 Sat

 248F-M1 R2 + +

 248F-M2 R2 ―

 273F-W R1 + + +

273F- 1 R1

 273F- 2 R1

 Proximity '' 175F-W R4 + + +

 175F-M R4 ―

 213F-W R3 + + +

213F-M R3 ―

 245F-W R2 + + +

245F-M1 R2 ―

 245F-M2 R2 ―

 248F-W R2 + + +

248F-M1 R2 +

 248F-M2 R2 ―

 273F-W R1 + + +

273F-M1 R1 ―

 273F-M2 R1 ―

 Remote part 175F-W R4 + + +

 175F-M R4 ―

 213F-W R3 + + +

213F- R3 ―

 245F-W R2 + + +

245F-M1 R2 ―

 245F- 2 R2 ―

 248F-W R2 + + +

248F-M1 R2 ―

 248F-2 R2 ―

 273F-W R1 + + +

273F-M1 R1 ―

273F-M2 R1 ― Table 2: Results of amplification reaction for case 2

 Yarn and Weave Forward Reverse Amplification Result Primer Primer

 Central focus 175F-W R4 + + +

 175F-M R4 ―

 213F-W R3 + + +

213F-M R3 ―

 245F-W R2 + +

 245F- 1 R2 + +

 245F- 2 R2 ―

 248F-W R2 + + +

248F- 1 R2 ―

 248F-M2 R2 ―

 273F-W R1 + + +

273F-M1 R1 ―

 273F-M2 R1 ―

 Proximity 175F-W R4 + + +

 175F-M R4 ―

 213F-W R3 + + +

213F-M R3 ―

 245F-W R2 + + +

245F-M1 R2 +

 245F-M2 R2 ―

 248F-W R2 + + +

248F-M1 R2 ―

 248F-M2 R2 ―

 273F-W R1 + + +

273F-M1 R1 ―

 273F-M2 R1 ―

 Transition 175F-W R4 + + +

 175F-M R4 ―

 213F-W R3 + + +

213F-M R3

 245F-W R2 + + +

245F-M1 R2 ―

 245F-M2 R2 ―

 248F-W R2 + + +

248F-M1 R2

 248F-M2 R2 +

 273F-W R1 + + +

273F-M1 R1 ―

273F-M2 R1 ― Table 2: Results of amplification reaction for case 2 (continued)

[0082] In Case 1, a point mutation was found at the 248th codon (codon encoding arginine) in the center of the cancer lesion, and a similar mutation was found in the vicinity of the lesion. It was thought that there was invasion of cancer cells to the edge. No mutations indicating cancer cells were observed in the remote area.

 [0083] In Case 2, the force of the point mutation was found in the 245th codon (codon encoding glycine) in the immediate vicinity of the main lesion. This mutation was not detected, and a strong mutation was found at codon 248. This suggests that the cancer tissue of the metastatic lesion is a cancer cell with a different origin from the metastasis of the primary focal force.

Claims

The scope of the claims

 [1] A method for detecting in a second specimen a cell related to a disease contained in the first specimen of subject power or a cell derived therefrom,

 (a) detecting a genetic mutation associated with the disease in the genome contained in the first specimen;

 (b) a step of detecting the genetic mutation in the genome contained in the second specimen, and

 (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation

 When the detected gene mutations are identical to each other, it is indicated that the disease-related cells contained in the first specimen or cells derived therefrom are detected in the second specimen force. Method.

 [2] The method according to claim 1, wherein the first specimen is collected before the treatment of the disease, and the second specimen is collected during or after the treatment of the disease.

 [3] The method according to claim 2, wherein the treatment is selected from the group consisting of drug therapy, radiation therapy, hyperthermia, hormone therapy, immunotherapy, and surgical therapeutic power.

 [4] The method according to claim 1, wherein the first specimen is obtained by collecting the force of the lesion of the disease, and the second specimen is obtained by collecting another force of the part.

 5. The method according to claim 1, wherein the disease-related cell is a cancer cell or a carcinoma cell.

 [6] The method according to claim 1, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.

 [7] The method according to claim 6, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.

 [8] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. In addition, in the target nucleic acid sequence, a sequence (ハ イ ブ リ ′) that hybridizes to a complementary sequence (Be) of the sequence (B) existing 5 ′ to the sequence (A) is represented by 5 in the sequence (Ac). The method according to claim 7, comprising:

[9] At least the primer set used in the nucleic acid amplification method can amplify the target nucleic acid sequence. A primer set comprising two kinds of primers,

 The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. The sequence comprises a sequence (Β ') that hybridizes to the complementary sequence (Be) of the sequence (B) existing 5' to the sequence (A) on the 5 'side of the sequence (Ac). A second primer included in the primer set, a sequence (Cc ') that hybridizes to a sequence (C) of the 3' end portion of the complementary sequence of the target nucleic acid sequence, and 3 at the end portion. And a folded sequence (D-Dc) containing two nucleic acid sequences that are mutually and non-hybridized on the same strand, on the 5 'side of the sequence (Cc). Method.

 [10] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence. 10. The method of claim 9, further comprising:

 [11] The method according to claim 7, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.

 [12] The method according to claim 7, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.

[13] The method according to claim 12, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.

[14] The method of claim 7, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.

 [15] A method for assessing the effect of treatment of a disease in a subject comprising:

 (a) Subject power The genome contained in the first sample collected before treatment of the disease! Detecting a genetic mutation associated with the disease,

 (b) subject power a step of detecting the gene mutation in a genome contained in a second specimen collected during or after treatment of the disease, and

 (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation

And the detected genetic variation is different from each other or in the second specimen If the gene mutation is not detected, the method is evaluated as having a high therapeutic effect.

 [16] The method of claim 15, wherein the treatment is selected from the group consisting of drug therapy, radiation therapy, hyperthermia, hormonal therapy, immunotherapy, and surgical therapeutic power.

 [17] The method according to claim 15, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.

 [18] The method according to claim 17, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.

 [19] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. In addition, in the target nucleic acid sequence, a sequence (ハ イ ブ リ ′) that hybridizes to a complementary sequence (Be) of the sequence (B) existing 5 ′ to the sequence (A) is represented by 5 in the sequence (Ac). 19. The method of claim 18, wherein the method comprises:

 [20] The primer set used in the nucleic acid amplification method is a primer set comprising at least two kinds of primers capable of amplifying the target nucleic acid sequence,

 The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. The sequence comprises a sequence (Β ') that hybridizes to the complementary sequence (Be) of the sequence (B) existing 5' to the sequence (A) on the 5 'side of the sequence (Ac). A second primer included in the primer set, a sequence (Cc ') that hybridizes to a sequence (C) of the 3' end portion of the complementary sequence of the target nucleic acid sequence, and 3 at the end portion. 19. A folded sequence (D-Dc) comprising two nucleic acid sequences that are mutually and non-hybridized on the same strand on the 5 ′ side of the sequence (Cc). Method.

 [21] The primer set is a third primer that hybridizes to the target nucleic acid sequence or its complementary sequence, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or its complementary sequence 21. The method of claim 20, further comprising:

[22] The method according to claim 18, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.

[23] The method according to claim 18, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.

[24] The method according to claim 23, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.

[25] The method according to claim 18, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.

 [26] A method for evaluating the primary focus of disease in a subject to metastasis to another site,

 (a) the primary focal force of the disease of the subject in the genome contained in the collected first specimen, detecting a genetic mutation associated with the disease,

 (b) another site force of the subject, detecting the gene mutation in the genome contained in the second sample collected, and

 (c) Step of comparing the first specimen force detected gene mutation and the second specimen force detected gene mutation

 And wherein the detected genetic mutation is identical to each other, the method is evaluated as having metastasis from the primary lesion site to another site.

[27] The method of claim 26, wherein the disease is cancer or tumor.

[28] The method according to claim 26, wherein the detection of the gene mutation is performed by a nucleic acid amplification method.

[29] The method according to claim 28, wherein the nucleic acid amplification method enables amplification of a target nucleic acid by a reaction under isothermal conditions.

[30] The first primer included in the primer set used in the nucleic acid amplification method comprises a sequence (Ac) that hybridizes to the sequence (A) at the 3 'end of the target nucleic acid sequence, and 3 at the end. And a sequence present 5 'to the target nucleic acid sequence from the sequence (A)

30. The method according to claim 29, comprising a sequence (Β ′) that hybridizes to the complementary sequence (Be) of (B) on the 5th side of the sequence (Ac).

[31] The primer set used in the nucleic acid amplification method is at least capable of amplifying the target nucleic acid sequence.

A primer set comprising two kinds of primers,

The first primer included in the primer set comprises a sequence (Ac ′) that is to be hybridized to the sequence (A) at the 3 ′ end portion of the target nucleic acid sequence at the 3 ′ end portion, and the target nucleic acid sequence. Complementary sequence (Be) of sequence (B) existing 5 ′ from sequence (A) A sequence (Β ′) that hybridizes to the 5 ′ side of the sequence (Ac), the second primer included in the primer set, and the 3 ′ end of the complementary sequence of the target nucleic acid sequence. The sequence (Cc ') that hybridizes to the sequence (C) of the part 3 and the folded sequence (D-Dc) that contains two nucleic acid sequences on the same strand, which are included in the end part and mutually cross and hybridize. 30. The method according to claim 29, comprising 5 'of the sequence (Cc).

 [32] The primer set is a third primer that hybridizes to the target nucleic acid sequence or a complementary sequence thereof, and a third primer that does not compete with other primers for hybridization to the target nucleic acid sequence or the complementary sequence. 32. The method of claim 31, further comprising:

 [33] The method according to claim 29, wherein a polymerase having strand displacement ability is used in the nucleic acid amplification method.

 [34] The method according to claim 29, wherein the nucleic acid amplification method is performed in the presence of a melting temperature adjusting agent.

[35] The method according to claim 34, wherein the melting temperature adjusting agent is dimethyl sulfoxide, betaine, formamide or glycerol, or a mixture of two or more thereof.

[36] The method according to claim 29, wherein the nucleic acid amplification reaction is performed in the presence of a mismatch binding protein.