JP2004350563A - Variation analysis method and system therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and inexpensive variation analysis method suitable for use at clinical sites, and to provide a system therefor. <P>SOLUTION: The variation analysis method comprises the following procedure: By using a set of primers so designed that the 3'-terminals coincide with a to-be-detected specific site( e.g. SNP(single nucleotide polymorphism) site ), a short domain close to the specific site is selectively amplified. Further, by controlling the modification temperature in the nucleic acid amplification step, only the true amplification product is dissociated/amplified, and the sequence of the specific site is analyzed by detecting the resultant amplification product. Thus, the objective variation analysis can easily be carried out by a single PCR amplification through preventing a pseudopositive amplification. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００７６】
【配列表フリーテキスト】
配列番号１−人工配列の説明：センスプライマー
配列番号２−人工配列の説明：アンチセンスプライマー
配列番号３−人工配列の説明：センスプライマー
配列番号４−人工配列の説明：アンチセンスプライマー
配列番号５−人工配列の説明：ＰＣＲ増幅産物
配列番号６−人工配列の説明：ＰＣＲ増幅産物
【図面の簡単な説明】
【図１】図１は、ＢＡＭＰＥＲ法によるＳＮＰ解析方法の概要を示す図である。
【図２】図２は、本発明によるＳＮＰ解析方法の原理を示す図である。
【図３】図３は、本発明によるｐ５３エキソン８のＳＮＰ解析における、電気泳動結果を示すグラフである。
【図４】図４は、ｐ５３エキソン８のＳＮＰ解析において、異なる変性温度（８０℃及び９４℃）でＰＣＲを行った場合の電気泳動結果を示すグラフである。
【図５】図５は、本発明のＰＣＲ温度特性を示すグラフである。Ａは、変性温度と相対増幅量の関係を示すグラフであり、Ｂは、塩基長と解離温度の関係を示すグラフである。
【図６】図６は、本発明を用いた検体多量解析の結果を示すグラフである。
【符号の説明】
１…ＰＣＲ産物のセンス鎖、１’…ＰＣＲ産物のアンチセンス鎖
２…ｄＮＴＰとプライマー残査
３…センス鎖中のＳＮＰ部分
４…アンチセンス鎖中のＳＮＰ部分
５…ＳＮＰ検査用プローブ
６…伸長鎖
７…ピロリン酸
２１…ゲノムのセンス鎖、２１’…ゲノムのアンチセンス鎖
２２…ゲノムセンス鎖中のＳＮＰ部分、２２’…ゲノムアンチセンス鎖中のＳＮＰ部分
２３…ゲノムセンスに結合するＳＮＰ用プローブ
２４…ゲノムアンチセンスに結合するＳＮＰ用プローブ
２５、２６…ＳＮＰに対応する識別配列
２７、２８…ゲノムの配列とは非相補な配列部分
２９、３０…ゲノムの各鎖とＳＮＰ判定用プローブのハイブリッド
３１…ＰＣＲ産物
４１…Ａホモザイゴトート検体
４２…Ｔホモザイゴトート検体
４３…ヘテロザイゴート検体
４４、４５、４６…末端がＴのＳＮＰ検査用プローブでの電気泳動結果
４７、４８、４９…末端がＡのＳＮＰ検査用プローブでの電気泳動結果
５０、５１、５２、５３…ＰＣＲ産物のピーク
５４…プローブのピーク
６１…変性温度８０℃でのＰＣＲ結果
６２…変性温度９４℃でのＰＣＲ結果
６３…目的ＰＣＲ産物
６４…プローブ
６５…擬陽性増幅ピーク
７１…目的のＰＣＲ産物由来の電気泳動分離ピーク面積の相対値
７２…擬陽性増幅産物に関わるピーク面積の合計の相対値
８１…アレルＡの検体グループ
８２…ヘテロザイゴートの検体グループ
８３…アレルＴの検体グループ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a mutation analysis method and a system therefor. More specifically, the present invention relates to a method for analyzing a single nucleotide polymorphism (SNP) for comparing gene sequences between individuals or within a population, and a system for the method.
[0002]
[Prior art]
One of the important tasks of the post-genome study, which is the next study after the decoding of the human genome, is single nucleotide polymorphism analysis, which examines minute differences in the sequence of each individual. The totality of single nucleotide polymorphisms (SNPs) in humans means the "individuality" of an individual. SNPs appear at a frequency of 1 in 1500 bases in the human genome, and many of them are thought to affect susceptibility to disease, easiness of cure, susceptibility to drugs, or environmental stress response. I have. Therefore, as post-genome research progresses, differences in the mechanisms that cause the underlying disease, even for diseases with the same diagnosis and similar symptoms, will become apparent at the molecular level. And it is expected that individualization of medical treatment (customization), such as proper use of drugs taking into account the difference, will be possible. In addition, it is possible to determine the risk of susceptibility to an individual's disease (disease susceptibility), and by building a lifestyle to avoid the disease as necessary, prevent the disease, delay its onset, and detect it early. -It is thought that early treatment can be realized.
[0003]
An SNP is a gene sequence in a meiotic genome that is replaced by another base, which is genetically inherited by crossing. Therefore, when examining the sequence of a group consisting of many individuals, two types of sequences at specific sites of the gene appear at a certain frequency. SNPs are classified into five types according to their locations. SSNPs present in the translation region without alteration of the amino acid sequence, cSNPs with amino acid change or incapable of producing protein, rSNPs and iSNPs present in the regulatory region and possibly affecting gene expression, and other regions GSNP with little effect on gene expression.
[0004]
All SNPs are useful as polymorphism markers to find genes associated with the onset or exacerbation of disease, but important in the clinical field are cSNPs and rSNPs that are directly related to disease risk diagnosis and proper use of drugs. An estimate is made of the number of SNPs considered medically significant. There will be 3 to 6 million SNPs in a human genome consisting of 3 billion base pairs. As a result of the human genome project, it has been found that the number of human genes is approximately 26,000 to 28,000, and the average size is 1 kb (1,000 bases). For this reason, the area where the meaningful SNP exists is narrowed down to the range of 26 to 28 Mb. In protein translation from a gene, amino acids are determined by triplet codons, which are sequence combinations every three bases. Of these, even if the third base changes, it is only translated into the same amino acid or replaced with an amino acid having similar properties, so that the function of the protein is affected by only 2/3 of the total. Therefore, SNPs involved in the function of the protein will be in the range of approximately 17-19 Mb. If the frequency of appearance of SNPs is one in 1.5 kb, the number of SNPs involved in the function is 13,000 at the maximum.
Of these, the number of meaningful SNPs included in the analysis is one factor that determines the SNP analysis market in the medical field. That is, even if the cSNP and the rSNP are combined, there is a high possibility that SNPs having a useful value will be narrowed down to several thousands of the same level as the number of genetic diseases.
[0005]
Thus, SNPs that are industrially important are limited, and SNPs that are predicted to have a causal relationship with disease are being actively searched for. For the search, a method of analyzing a large amount of SNPs is required, and a number of methods and devices have been reported for the method. For example, as a method of searching for an SNP candidate from a completely unknown region, there is a conventional DNA sequencing method in which the sequence of a specific region is analyzed and compared with a plurality of samples. In addition, as a method for analyzing SNP candidates narrowed down to some extent, there are SSCP method utilizing the fact that the stabilization energy when DNA forms a higher-order structure differs by one base, and denaturant concentration gradient gel electrophoresis. (DGGE), heat denaturing HPLC method (WAVE (Registered trademark) system), a method of selectively chemically cleaving the SNP site of a chimeric duplex obtained by mixing a sample containing an SNP site (CCM), an S1 nuclease method for enzymatically cleaving the chimeric portion, a known method. Invader method for typing in SNP, TaqMan method, DNA chip method for comparing differences in hybridization stability with probes, mini-sequence method for determining local sequence of only SNP base sites (for example, see Non-Patent Document 1) A mass array method combining mini-sequence and mass spectrometry has been developed. Further, as an analysis method for the purpose of clinical testing of SNP, there is a BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions) method using biochemiluminescence (for example, see Patent Document 1 and Non-Patent Document 2).
[0006]
In the BAMPER method, an extension reaction is performed on a target gene using two types of probes designed so that the 3 ′ end of the target gene matches the SNP allele, and pyrophosphate generated at that time is converted into a luciferin-luciferase reaction. By biochemiluminescence. Since the extension reaction proceeds only when a mutation corresponding to the probe used is present in the target gene, the SNP sequence of the target gene can be determined in a few minutes by measuring luminescence. The BAMPER method is considered to be an analysis method suitable for use in a clinical setting in that it can perform SNP analysis only by adding a reagent and can be carried out with a simple optical system luminescence measuring device.
[0007]
However, SNP analysis by the BAMPER method requires four steps of reactions including two complementary strand synthesis reactions. Hereinafter, each step will be described with reference to FIG.
(1) PCR amplification of a portion consisting of several hundred bases including the SNP site to be detected:
This step aims not only to amplify the sample but also to amplify a limited region including the SNP site, thereby reducing the false positive amplification reaction in the subsequent SNP detection reaction. As a result, a mixture of the PCR product of the sense strand 1 having the SNP site 3 and the antisense strand 1 'having the portion 4 complementary to the SNP site, dNTP which is the reaction residue 2, and the primer are obtained.
[0008]
(2) Purification of PCR product from PCR reaction solution:
In the BAMPER reaction, since pyrophosphate generated by the probe extension reaction is detected, the PCR residue 2 composed of primers and dNTP used in PCR interferes with the measurement. That is, the PCR primer hybridizes to the PCR product, causes complementary strand synthesis by the DNA polymerase, and generates pyrophosphate. In addition, dNTP is a pseudo substrate for a luminescence reaction for detecting pyrophosphate. Therefore, in order to remove these, in FIG. 1, the PCR residue 2 is decomposed with Shrimp alkaline phosphatase and Exonuclease I. In the first report of the BAMPER method (Non-Patent Document 2), PCR amplification was performed using biotin-labeled primers, and the amplified products were captured on magnetic beads with streptavidin to remove extra PCR primers and dNTPs. . The supplemented PCR product is denatured with alkali, so that only the strand immobilized on the beads can be used as a template for the next BAMPER extension reaction.
[0009]
(3) Preliminary stage of BAMPER reaction (complementary strand extension reaction using BAMPER probe):
The purified template PCR product is hybridized with a BAMPER probe having a structure in which the 3 'end of probe 5 is complementary to the SNP site and having a length of about 20 to 30 bases, and a complementary strand elongation reaction is performed by the catalytic action of DNA polymerase. Let Here, in order to increase the fidelity of the probe extension reaction corresponding to the SNP sequence, a base sequence that is mismatched with the sample DNA sequence is inserted in advance into the third to fourth bases from the 3 ′ end of the probe. (For example, see Non-Patent Documents 2 and 3).
[0010]
Only when the probe has a sequence complementary to the SNP site at the 3 'end, an extension reaction takes place, producing 6 extended chains and pyrophosphate 7. Since one molecule of pyrophosphate is generated per one base extension, the amount of pyrophosphate increases with the extension reaction. Probes with non-complementary 3 'ends do not undergo extension reactions and do not produce pyrophosphate. In general, the reaction cycle repeats a denaturation step at 94 ° C. (10 seconds) and a hybridization and extension reaction step at 50 to 60 ° C. (10 seconds) about five times in order to increase the amount of generated pyrophosphate. Since the length of the DNA extension chain in the BAMPER reaction is at most about 200 bases, the extension reaction occurs sufficiently even in a short reaction cycle.
[0011]
{Circle around (4)} After stage of BAMPER reaction (quantitative detection of pyrophosphate by luminescence reaction):
Finally, pyrophosphate 7 generated by the extension reaction of the BAMPER probe is converted to ATP, and detected by a luminescence reaction using luciferin-luciferase. That is, light emission at around 530 nm, which is generated when oxidized luciferin in an excited state generated by the action of ATP and luciferase returns to the ground state, is detected. Since only the BAMPER probe complementary to the SNP sequence can emit light, the sequence of the SNP can be determined based on the sequence at the 3 'end of the BAMPER probe that has emitted light.
[0012]
[Patent Document 1]
JP-A-2002-101899
[Non-patent document 1]
A. Jalanko, et al. , Clinical Chemistry, (1992), 38, p39-43.
[Non-patent document 2]
Guo-hua Zhou, et al. , Nucleic Acid research, (2001), 29, e93.
[Non-Patent Document 3]
K. Okano, et al. , Electrophoresis, (1998), 19, p3071-3078.
[0013]
[Problems to be solved by the invention]
As described above, SNP analysis by the BAMPER method requires four steps of reactions including two complementary strand synthesis reactions. If this can be performed in fewer steps, the operation is expected to be simpler and the automation of the process is expected to be easier.
[0014]
That is, an object of the present invention is to provide a simple and inexpensive SNP analysis method and a system therefor which are improved in SNP analysis technology based on the BAMPER method and are more suitable for use in clinical settings.
[0015]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the inventors have found that in the SNP analysis by the BAMPER method, the PCR amplification of (1) and the complementary strand extension reaction by the BAMPER probe of (3) are both complementary by DNA polymerase. It was noted that this was a chain synthesis reaction. Then, it was thought that if one of the primers used for PCR was designed so that the 3 'end coincides with the SNP site as in the case of the BAMPER probe, the complementary strand elongation reaction using the BAMPER probe could be performed together with PCR.
[0016]
However, in practice, false positive amplification occurs due to nonspecific hybridization of primers, so that it was difficult to specifically detect the SNP sequence only by the above method. Therefore, the present inventors conducted further studies, and by devising primers and PCR conditions, selectively amplify a short region near the SNP and detect the SNP sequence by one PCR without causing false positive amplification. I found a way to do it.
[0017]
That is, the present invention is a nucleic acid amplification method for obtaining an amplification product of a desired base length range including a specific site on a target nucleic acid,
A first primer comprising a sequence complementary to the base sequence of the first strand of the target nucleic acid and having a 3 ′ end designed to hybridize to the specific site on the first strand; and a second primer of the target nucleic acid. Performing nucleic acid amplification using a second primer containing a sequence complementary to the base sequence of the strand and having a 3 ′ end designed to hybridize to a site forming a base pair with the specific site on the second strand. ,as well as
Controlling the denaturation temperature in the nucleic acid amplification to dissociate the amplification product in the desired base length range into a single strand and use it as a template for the next nucleic acid synthesis reaction. provide.
[0018]
In the above method, the denaturation temperature in nucleic acid amplification is initially set as usual high (for example, around 94 ° C.) to proceed with the amplification reaction, and is lowered to 60 to 85 ° C. when the amount of short-chain amplification products from the primers increases to some extent. Control. Specifically, it is preferable to control the temperature to be reduced to 60 to 85 ° C. from at least the fifth cycle. As a result, only the short-chain nucleic acid, which is a true amplification product from the primer, is dissociated and becomes a template for the next nucleic acid synthesis reaction, so that false positive amplification due to non-specific hybridization of the primer is prevented.
[0019]
In the present invention, an amplification product having a desired base length range is a nucleic acid synthesized by hybridizing the 3 'end of the first and second primers to a specific site on the target nucleic acid. Therefore, the desired base length range is a value defined by the base lengths of the first primer and the second primer (base length obtained by subtracting 1 from the sum of the base lengths of both primers).
[0020]
The first primer and the second primer preferably have a base length of 18 to 50 bases and have substantially the same melting temperature. In addition, the sum of the ratios of guanine and cytosine in the first primer and the second primer is desirably 35 to 65%.
[0021]
The present invention also provides a method for analyzing the sequence of a specific site on a target nucleic acid. The method comprises the steps of: a first primer comprising a sequence complementary to a base sequence of a first strand of the target nucleic acid, and a first primer designed to hybridize to the specific site on the first strand at a 3 ′ end; A nucleic acid using a second primer which contains a sequence complementary to the base sequence of the second strand of the nucleic acid and whose 3 ′ end is designed to hybridize to a site forming a base pair with the specific site on the second strand; Performing amplification; and
By controlling the heating temperature in the nucleic acid amplification, the amplification product in the desired base length range is dissociated into single strands, and a nucleic acid amplification step characterized by being used as a template for the next nucleic acid synthesis reaction; and
Analyzing the sequence of the specific site on the target nucleic acid by detecting the amplification product.
[0022]
Here, the specific site to be detected includes, for example, a single nucleotide polymorphism site.
As a method for detecting an amplification product, any known method such as electrophoresis may be used.However, a method for detecting pyrophosphate generated in response to nucleic acid amplification by a biochemiluminescence reaction requires an apparatus or an operation. It is preferable in that it is simple.
[0023]
The present invention also provides a system for the analysis method. The system includes a reaction vessel containing a reaction solution containing a target nucleic acid and a primer for obtaining an amplification product in a desired base length range including a specific site on the target nucleic acid,
A temperature control mechanism for controlling the temperature in the reaction vessel to dissociate and amplify the amplification product in the desired base length range,
Having a light detection mechanism for detecting light emission generated in the reaction tank, and
The sequence of the specific site contained in the target nucleic acid is automatically analyzed by recognizing the type of the primer that has generated the biochemiluminescence.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
The SNP analysis method of the present invention comprises three steps of (1) PCR amplification, (2) purification, and (3) SNP-specific complementary strand extension reaction required by the BAMPER method by devising primers and PCR conditions. The process is performed by a single operation to simplify and speed up the operation.
[0025]
In particular, the present invention has a great feature in that by devising the above primers and PCR conditions, the problem of false positive amplification, which is a problem when detecting nucleic acids, is overcome. Hereinafter, the present invention will be described in detail.
[0026]
1 primer
1) Function of primer
In the present invention, a short region in the vicinity of a specific site is selectively amplified by using a primer set designed such that its 3 'end is coincident with a specific site (for example, an SNP site) to be detected. At the same time, since the primer also functions as a BAMPER probe, the sequence of a specific site in the target nucleic acid can be analyzed only by PCR amplification.
[0027]
In the present invention, the selectivity for the sequence to be detected is improved by designing the primers corresponding to the sense strand and the antisense strand so that the 3 'ends of the primers coincide with the SNP site. That is, even if false positive amplification occurs in which the primers are non-specifically hybridized to the genome, the smallest product in the PCR product is the true amplification product of interest. Therefore, false positive amplification can be prevented by controlling the denaturation temperature during PCR amplification described below to selectively dissociate the short-chain true amplification product and using it as a template for the next nucleic acid synthesis reaction. . In the present specification, the “true amplification product” means an amplification product synthesized by hybridizing the 3 ′ ends of the first primer and the second primer to a specific site on the target nucleic acid. .
[0028]
2) Primer set
The primer includes a sequence complementary to the base sequence of the first strand of the target nucleic acid, and a first primer designed to hybridize the 3 ′ end to a specific site on the first strand; A pair of primers comprising a second primer designed to hybridize to a site that forms a base pair with the specific site on the second strand, the second primer comprising a sequence complementary to the base sequence of the two strands; Be composed. In the case of single nucleotide polymorphism detection, at least two primer sets are required corresponding to each allele, and in some cases, three or four primer sets are required. That is, the sequences at the 3 'end of the primer include A / C group, A / G group, A / T group, C / G group, C / T group, G / T group, A / C / G group, and A A / C / T group, A / G / T group, C / G / T group and A / C / G / T primer group are conceivable.
[0029]
3) Introduction of mismatch
In the above primer, a structure (mismatch) different from the sequence of the corresponding genomic sample is introduced at the position of the 3rd to 4th bases 5 'from the 3' end of at least one primer, preferably both primers. Is preferred. Thereby, the sequence specificity of the mutation site (for example, SNP allele) of the amplification reaction in PCR is remarkably increased. In the conventional BAMPER method, similar mismatches are introduced into primers.However, in normal PCR, primers are designed so as to hybridize to distant positions, so that only one primer is artificially non-complementary. No site (mismatch) can be introduced. Since the primer of the present invention has a structure in which the 3 'ends overlap, it is possible to introduce a mismatch into both primers. This significantly improves the accuracy of the analysis.
[0030]
In PCR using a primer set in which the 3 'end matches the SNP position, there is no degree of freedom in designing PCR primers, and the sequence position is almost uniquely determined. For this reason, when palindrome appears in the primer, the primer itself often has a higher-order structure, and the hybridization to the template DNA is often inhibited. In order to avoid this, when at least one of the primers has a structure such as 4 bases, 6 bases, or 8 bases that forms a palindrome, any one of the halves from the 5 ′ end of the sequence site that forms the palindrome is used. The palindromic structure is eliminated by setting one base different from the original sequence.
[0031]
4) Base length
The primer used in the present invention has a length of 18 to 50 bases, particularly about 18 to 30 bases, as in the case of ordinary PCR primers.
In the present invention, both the 3 'ends of both primers are designed to hybridize to a specific site (SNP site or the like) on the target nucleic acid. Therefore, the base length of the “true amplification product” synthesized by hybridizing the 3 ′ end of the first primer and the second primer to the specific site on the target nucleic acid is the same as that of the first primer and the second primer. Is defined by the base length of the primer. That is, the base length of the true amplification product is “the base length obtained by subtracting 1 from the sum of the base lengths of the first primer and the second primer”. For example, when a primer set having a length of 18 to 50 bases is used, a desired base length range is 35 to 99 bases.
[0032]
5) Melting temperature (Tm)
In the present invention, the first primer and the second primer preferably have substantially the same melting temperature (Tm). Here, “substantially the same” means that they are not completely the same in terms of calculation, but the difference is slight, about 5 ° C. or less, and can be treated as substantially the same Tm in the experiment. I do.
[0033]
6) GC content (%)
In addition, the sum of the ratios (GC content (%)) of guanine and cytosine in the first primer and the second primer is preferably 35% to 65%.
In the present invention, the GC content (%) of the true amplification product is almost equal to the sum of the GC content (%) of both primers. Although the Tm of a nucleic acid varies depending on its base length and GC content (%), as will be described later, a short chain having a base length of about 35 to 99 bases (35 to 99 bases) at a denaturation temperature of 60 to 85 ° C. without causing false positive amplification. In order to selectively dissociate the true amplification product (about the base length), the GC content (%) of the amplification product is preferably 35% to 65%, particularly preferably 40% to 60%. Therefore, the sum of the GC contents (%) of both primers is preferably 35% to 65%, particularly preferably 40% to 60%.
[0034]
2. PCR conditions
Another feature of the present invention resides in temperature control during PCR amplification. In the nucleic acid amplification step, as in a normal PCR reaction cycle, it is necessary to sequentially change the temperature to a denaturation temperature for dissociating the template nucleic acid into single strands, a temperature for annealing, and a temperature for extension reaction. is there. In particular, in the case of the present invention, the denaturation temperature in nucleic acid amplification is initially high as usual, for example, set at 90 to 100 ° C., preferably around 94 ° C., and the amplification reaction proceeds. Is controlled to lower the denaturation temperature to 60-85 ° C. Specifically, it is preferable to control the temperature at 60 to 85 ° C, preferably 80 to 85 ° C, from at least the fifth cycle. As a result, only the short true amplification product can be dissociated, and even if false positive amplification occurs, the product will not be used as a template for the next nucleic acid synthesis.
[0035]
Of course, the denaturable temperature of the PCR product varies depending on the base length and GC content (%) of the extension product. For example, when the GC content (%) is 58%, Tm is 71 ° C. for a PCR product having a length of 60 bases, 76 ° C. for a length of 80 bases, 77 ° C. for a length of 100 bases, 79 ° C. for a length of 150 bases, and 79 ° C. for a length of 200 bases. 81 ° C. (see FIG. 5B). Substantially the denaturation temperature of the PCR needs to be even higher by 5-10 ° C. than these Tm values. Therefore, when the base length of the PCR product is 80 bases, the denaturation temperature is preferably from 80 to 85 ° C. (see FIG. 5B). In other words, when the GC content (%) is 58%, the PCR product that can be amplified at the denaturation temperature of 80 ° C. has a limit of 80 bases in length.
[0036]
On the other hand, if the GC content (%) decreases, the Tm value also decreases, and the preferable denaturation temperature also decreases (the base length of the PCR product that can be amplified at the denaturation temperature of 80 ° C. increases). Further, when the GC content (%) increases, the Tm value also increases, and the preferable denaturation temperature also increases (the base length of the PCR product that can be amplified at the denaturation temperature of 80 ° C. decreases).
[0037]
The true amplification product of the present invention has a length of 35 to 99 bases when a primer having a length of 18 to 50 bases is used. Denaturation becomes possible in the range of 85 ° C.
In the present invention, if necessary, a Tm adjuster or the like may be used to adjust the Tm of the amplification product.
[0038]
3. Detection method
In the present invention, the amplification reaction proceeds only when a primer set corresponding to the sequence of the specific site of the target nucleic acid is used. That is, the sequence at the specific site can be determined based on the sequence at the 3 'end of the primer set that has generated the amplification product.
[0039]
In the present invention, the detection of a desired true amplification product may be performed by any known detection method such as electrophoresis, fluorescence detection using energy transfer, and detection using biochemiluminescence described below. The method is preferred in that the device and operation are simple.
[0040]
In PCR, when one high-energy triphosphate bond is cleaved, one inorganic pyrophosphate is released. That is, one pyrophosphate is generated by the extension reaction of one base. Thus, by detecting this pyrophosphate, it is possible to indirectly detect an amplification product.
[0041]
Pyrophosphate is detected using biochemiluminescence with luciferin-luciferase, as in the BAMPER method. That is, first, it is converted to ATP using pyrophosphate diphosphokinase or ATP sulfurylase. When the converted ATP and luciferin are oxidatively reacted with luciferase as an enzyme, oxyluciferin, pyrophosphate, and a series of other substances are produced. Light emission in the vicinity of 530 nm generated when the generated luciferin in the excited state returns to the ground state is detected. Since luminescence can be generated only by a primer complementary to the sequence of a specific site (for example, an SNP site) on the target nucleic acid, the sequence of the specific site can be determined by the sequence at the 3 'end of the primer that generated luminescence.
[0042]
In the biochemiluminescence using luciferin-luciferase, the sample must not contain ATP or dATP which is a substrate for pyrophosphate or luciferase, and the short chain which causes a non-specific complementary strand synthesis reaction. DNA or single-stranded part of the DNA. Therefore, pyrophosphate, ATP, and dATP contained in the sample are decomposed in advance by phosphatase, and short-chain DNA and single-stranded portions are decomposed by exonuclease I.
[0043]
4. Mutation analysis system
When the method of the present invention is carried out using biochemiluminescence, separation and analysis means such as electrophoresis and chromatography are unnecessary, and there is no need for excitation with a laser or the like, which is suitable for automation of the operation. . The present invention provides a system for such automatic analysis.
[0044]
For example, the system includes a reaction vessel containing a reaction solution containing a target nucleic acid and a primer for obtaining an amplification product in a desired base length range including a specific site on the target nucleic acid, and controlling the temperature in the reaction vessel. A temperature control mechanism for dissociating and amplifying the amplification product in the desired base length range, and a light detection mechanism for detecting luminescence generated in the reaction vessel, and wherein the biochemiluminescence is generated. The type of the primer is recognized, and the sequence of the specific site contained in the target nucleic acid is automatically analyzed.
[0045]
The temperature control mechanism for dissociating and amplifying the amplification product in the desired base length range is a temperature control mechanism for a normal PCR reaction cycle (denaturation temperature for dissociating a template nucleic acid into a single strand, annealing temperature, etc.). And a temperature for the extension reaction, a mechanism for sequentially changing the temperature in the reaction vessel), and at the same time, the denaturation temperature in nucleic acid amplification is initially set as usual high (for example, around 94 ° C.), Means a control mechanism that lowers the denaturation temperature to 60-85 ° C. if the amount of short-chain true amplification products from E. coli increases to some extent (eg, after the fifth cycle).
The above system can be suitably used for mutation analysis such as SNP analysis at a clinical site.
[0046]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.
[0047]
Example 1: Mutation analysis of p53 exon 8 using electrophoresis
The mutation present in p53 exon 8 was regarded as a SNP, amplified using the method of the present invention, and analyzed by electrophoresis.
[0048]
1. Equipment and samples
As the thermal cycler, DNA Engine PTC200 (MJ Research) was used. For confirmation of the PCR product, a microchip electrophoresis analysis system SV1210 (Hitachi Electronics Engineering) was used. Wallac 1420 ARVOsx (PerkinElmer) was used as a luminometer. Primer synthesis was outsourced to SIGMA.
[0049]
A human-derived genome was used as a sample. The genome was hemolyzed by adding a 9-fold volume of 50 mM NaCl solution to 2 ml of blood, centrifuged at 3500 G, suspended in 10 ml of 50 mM NaCl solution, and centrifuged again to obtain a precipitate containing leukocytes. 1 ml of DNAzol was added to the precipitate to lyse leukocytes, and insoluble components were removed by centrifugation. An equal volume of butanol was added thereto and centrifuged. The precipitate was redissolved in 100 ml of water, and low molecular impurities were removed by an ethanol precipitation method. Finally, the sample was dissolved in sterilized water so that the genome concentration became 2.5 to 25 ng / μl, and used as a sample genomic solution. In the prepared sample genome, SNP alleles were identified in advance, and two types of homozygotes (AA), (TT), and heterozygotes (AT) were used in the following experiments.
[0050]
2. Method
This example is described using the region of p53 exon 8 shown in FIG. 21 represents the sense strand of p53 exon 8, and 21 'is the antisense strand. 22 (22 'in the case of the complementary strand) is the target displacement site. Although this part is not a SNP but a mere mutated part of the sequence, it is used as a model to explain the present invention. In the conventional BAMPER method, for example, the extension reaction is performed only with the primer 24 complementary to the antisense strand 21 ', but in the present invention, the primers 23 and 24 are used.
[0051]
The two primers used were such that the primer 24 was substantially complementary to the double-stranded antisense strand 21 ′ of the genomic sample, and the other primer 23 was substantially the sense strand 21 that was the other strand of the genome. Is complementary to The 3'-ends 25 and 26 of both primers are complementary to the bases of alleles 22 and 22 'of the SNP to be detected, respectively. This time, the SNP to be detected is a portion corresponding to Cys275Ser present in p53 exon 8.
This is a polymorphism at the 275th codon in the amino acid sequence, and the allele of the nucleotide sequence is A / T. Therefore, two sets of the above primer sets (23 and 24) were prepared, one for detecting an allele having an SNP site of A and another for detecting an allele having a SNP site of T.
[0052]
Furthermore, sequences 27 and 28 which are non-complementary to p53 exon 8 (sample DNA) were introduced into the third base from the 3 'end of each primer to enhance selectivity. That is, the third base from the 3 'end of the primer complementary to the antisense strand was changed from T corresponding to p53 exon 8 original sequence to A. The primer complementary to the sense strand was changed from A to T corresponding to the original sequence. As a result, in the primer set in which the 3 'end of the primer is complementary to the allele, two bases out of the three bases near the 3' end of the primer become completely complementary and the complementary strand elongation by DNA polymerase occurs, but the 3 'end is not complementary to the allele. In such a primer set, the 3 ′ end and the second base of the third base are non-complementary, so that the vicinity of the 3 ′ end of the primer cannot hybridize to the template DNA, and extension of the complementary strand by the DNA polymerase hardly occurs.
[0053]
The primer 23 was designed to be 20 bases long and the primer 24 to be 22 bases long so that the Tm at the time of PCR was 54.5 ° C. and 55.4 ° C. These values were calculated at a salt strength of 50 mM when PCR was actually performed. The optimum temperature during PCR hybridization (at the time of annealing) is 47.2 ° C., but this experiment was performed at 50 ° C.
[0054]
The sequences of the two primer sets used are shown below.
Primer set for detecting A allele at SNP site
sense primer: 5'-aac agc ttt gag gtg cgt gAt T-3 '(SEQ ID NO: 1, 22 bases long)
antisense primer: 5'-tct ctc cca gga cag gcT cA-3 '(SEQ ID NO: 2, 20 bases long)
Primer set for detecting T allele at SNP site
sense primer: 5'-aac agc ttt gag gtg cgt gAt A-3 '(SEQ ID NO: 3, 22 bases long)
Antisense primer: 5'-tct ctc cca gga cag gcT cT-3 '(SEQ ID NO: 4, 20 bases long).
[0055]
The PCR product obtained with the above primers is 41 bases long (bp), and the one corresponding to allele A is 5′-aac agc tt gag gtg cgt gAt TgA gcc tgt cct ggg aga ga-3 ′ (SEQ ID NO: 5). The one corresponding to allele T is 5′-aac agc ttt gag gtg cgt gAt AgA gcc tgt cct ggg aga ga-3 ′ (SEQ ID NO: 6). Their Tm is calculated to be 65.4 ° C.
[0056]
2 μl of a genomic DNA sample prepared at 2.5 to 25 ng / μl was added to a 96-well PCR plate, and placed on ice. 5 unit / μl Taq. 0.05 μl of DNA polymerase (manufactured by QIAGEN), 1 μl of 2.5 mM dNTPs, and 1 μl of a primer set corresponding to each allele of 25 pmol / μl (control uses sterile water) are mixed and sterilized. Water and Buffer for PCR were added to prepare a final volume of 23 μl per well. In practice, Taq. It is more efficient and more reproducible for PCR to prepare a reaction solution containing a DNA polymerase, dNTPs and a primer set in advance for one sample more than the required amount, and to add 23 μl of this to each well. In addition, about each genomic DNA sample, what added sterilized water instead of the primer set as a control was similarly used for reaction.
[0057]
Next, the upper part of the PCR plate was sealed with an adhesive sheet, and a PCR cycle reaction was performed. In PCR, the genome was first denatured by heating at 94 ° C. for 10 seconds, and then a cycle of 50 ° C. for 10 seconds and 80 ° C. for 10 seconds was repeated 35 times.
[0058]
3. result
FIG. 3 shows the results of performing PCR using two sets of primers corresponding to the SNP sequence and analyzing by electrophoretic separation. 41, 42, and 43 are the results obtained from the genomes of homozygote (A / A), homozygote (T / T), and heterozygote (A / T), respectively. In the figure, reference numerals 44, 45, and 46 denote electrophoretic separation patterns when a PCR reaction was performed using a primer set whose allele was complementary to A, and 47, 48, and 49 were primer sets whose allele was complementary to T.
[0059]
From the homozygote (A / A) genome, a specific amplification peak 50 was obtained only when amplification was performed with a primer set complementary to A. From the homozygote (T / T) genome, a specific amplification peak 51 was obtained only when amplification was performed with a primer set complementary to T. On the other hand, from the heterozygous (A / T) genome, specific amplification peaks 52 and 53 were obtained when the amplification was performed with either the primer set complementary to A or the primer set complementary to T. The peak 54 commonly detected in all the electrophoresis patterns corresponds to a probe.
[0060]
In this PCR system, since the amplified base length is as short as 41 bases, the temperature cycle may be as short as 50 ° C. for 10 seconds and 80 ° C. for 10 seconds. Further, since the denaturation temperature is as low as 80 ° C., there is an advantage that the time required for raising and lowering the temperature can be reduced. Actually, when the time required for PCR is calculated by assuming a temperature rise and fall rate of 3 ° C./sec, a rise from 55 ° C. to 80 ° C. per cycle takes about 10 seconds, a cycle at 80 ° C. for 10 seconds, a fall from 80 ° C. to 55 ° C. Takes about 10 seconds and 40 seconds at 55 ° C. for 10 seconds. In other words, the entire process is completed in about 20 minutes. In the case of this example, since the time required for microchip electrophoresis was about 3 minutes, it was confirmed that SNP determination (typing) from the genome could be performed in a short time even from PCR to detection.
[0061]
Example 2: Examination of PCR conditions
Using the primers and genome corresponding to the allele shown in Example 1, the denaturation temperature dependence of the false positive amplification reaction was examined.
[0062]
FIG. 4 shows the electrophoretic separation pattern of the amplified product when the higher temperature in the PCR cycle was 94 ° C., which is the same as that of ordinary PCR, and the temperature was 80 ° C. used in the present invention. 63 is the target PCR amplification product. 64 is a peak derived from the probe. A series of peaks 65 appearing in the pattern 61 depend on the pseudo-yang amplification. That is, it was confirmed that false positive amplification can be prevented by lowering the higher temperature of the PCR to 80 ° C.
This is because the PCR product generated with the primer of the present invention is a short chain, so that the amplification product can be sufficiently denatured even at a low temperature of about 85 ° C.
[0063]
In order to further verify this, the same experiment was performed at the denaturation temperature (higher temperature) of PCR at 75 ° C., 80 ° C., 85 ° C., 90 ° C., 95 ° C., and 97 ° C., and the results of comparison are shown in FIG. 5A. . In the figure, the plot of 71 indicated by ● represents the relative value of the area of the electrophoresis separation peak 63 derived from the target PCR product. The plot 72 indicated by △ in the figure represents the relative value of the peak area related to the false positive amplification product 65 observed in FIG. Here, the relative value of the area is calculated assuming that the area value of the temperature when the peak area is the maximum in the peak series of 61 and 62 is 1.0.
[0064]
FIG. 5A shows that when the denaturation temperature is in the range of 80 to 85 ° C., the desired PCR product can be obtained, but the false positive amplification is below the detection limit. On the other hand, at 90 ° C. or higher, although the target product is obtained, the number of false positive amplifications is increased. Note that the amount of the product decreased at 97 ° C., because the enzyme was thermally denatured.
[0065]
Of course, the denaturable temperature of the PCR product varies depending on the base length and GC content (%) of the extension product. FIG. 5B shows the relationship between the base length of the extension product and the denaturation temperature. In this example, the calculated Tm is 65 ° C. when the base length of the PCR product is 41 bases. Assuming that the GC content (%) is 58%, Tm is 71 ° C. when the PCR product is 60 bases, 76 ° C. for 80 bases, 77 ° C. for 100 bases, 79 ° C. for 150 bases, and 81 ° C. for 200 bases. ° C. Substantially the denaturation temperature of the PCR needs to be even higher by 5-10 ° C. than these Tm values. Therefore, when the base length of the PCR product is 80 bases, the denaturation temperature is 60 to 85 ° C, preferably 80 to 85 ° C. Therefore, a PCR product that can be amplified at a denaturation temperature of 80 ° C. has a limit of 80 bases.
[0066]
Example 3: Mutation analysis of p53 exon 8 using biochemiluminescence
Using the sample genome and the primer set prepared in Example 1, the mutated portion corresponding to Cys275Ser present in p53 exon 8 was analyzed using biochemiluminescence with luciferin-luciferase. The allele of the SNP site to be detected is A / T as in Example 1.
[0067]
1. sample
In biochemiluminescence using luciferin / luciferase, the sample must not contain ATP or dATP as a substrate for pyrophosphate or luciferase, and short-chain DNA that causes a non-specific complementary strand synthesis reaction. And no single-stranded part.
Therefore, in this example, it is necessary to decompose pyrophosphate, ATP, and dATP contained in the genome prepared by the method of Example 1 with phosphatase in advance, and to decompose the short-chain DNA and the single-stranded portion with exonuclease I. is there.
[0068]
That is, in a well of a 384-well PCR plate, 2 μl of a genomic DNA sample prepared at 8 to 56 ng / μl, 1 μl of shrimp alkaline phosphate (SAP) at a concentration of 1 unit / μl, and 10 unit / μl of exonuclease I A mixture of 0.1 μl of (ExoI), 0.5 μl of 10 × PCR buffer (manufactured by Amersham Pharmacia) and 1.4 μl of sterilized water was added. The plate is spun down to collect the liquid on the wall. The volume at this point is 5 μl.
[0069]
2. PCR reaction
The upper part of the PCR plate was sealed with an adhesive sheet, and set on a thermal cycler. Since SAP and Exo I are vulnerable to heat, the heat lid of the thermal cycler was not used, and a 3 mm-thick silicon sheet was covered and a weight was placed thereon to insulate and insulate hermetically. After incubating at 37 ° C. for 40 minutes to carry out the enzyme reaction, the enzyme was inactivated by heating at 80 ° C. for 15 minutes, and the temperature was raised to 95 ° C. for 5 seconds to denature the genome, followed by rapid cooling on ice. .
[0070]
The genomic sample solution after the above pretreatment was dispensed by 1 μl into three wells of a PCR plate. 5 unit / μl Taq. 0.02 μl of DNA polymerase (QIAGEN), 0.4 μl of 2.5 mM dNTPs, 0.08 μl of each primer set corresponding to 25 pmol / μl allele (control uses sterile water), 0.8 ml of 10 × PCR buffer (manufactured by Amersham Pharmacia, containing 15 mM Mg ion) and 10 mM MgCl 2₂0.45 ml (final magnesium concentration at the time of PCR was 2 mM) and sterile water were added so that the solution per well became 9 μl. All the operations so far were performed on ice. In addition, about each genomic DNA sample, what added sterilized water instead of the primer set as a control was similarly used for reaction.
After 5 μl of mineral oil was layered on each well to prevent drying, a PCR cycle reaction was performed. In PCR, a cycle of 55 ° C. for 10 seconds and 80 ° C. for 10 seconds was repeated 35 times in total.
[0071]
3. Biochemiluminescence detection
In Example 1, PCR products were separated and analyzed by electrophoresis. In this example, PCR products were detected using biochemiluminescence. A luminescent reagent (Kikkoman Co., Ltd.), which had been returned to room temperature in advance, was added to each of the PCR-completed samples in a set of three wells in an amount of 18 μl, mixed by pipetting five times, and measured with a luminometer. The luminescent reagent manufactured by Kikkoman is a reagent for measuring pyrophosphate described in “Food Industry Vol. 44 No. 14 p25-34”. AMP and phosphoenolpyruvate (PEP) are added to pyrophosphate using pyruvate phosphodikinase. To produce ATP. Since this reagent uses luciferase derived from a heat-resistant recombinant Japanese Genji firefly, there is an advantage that the reagent is stable and quality control is easy.
[0072]
Allele determination was performed using Ia as the signal value obtained from the primer set complementary to Allele C, It as the signal value obtained from the primer set complementary to Allele T, and Ic as the signal value from the control containing no primer. The signal intensity ratio (Ia−Ic) / (Ia + It−2 × Ic) and the signal intensity ratio (It−Ic) / (Ia + It−2 × Ic) derived from allele T were calculated. When the criterion is (Ia−Ic) / (Ia + It−2 × Ic) <0.2 (or (It−Ic) / (Ia + It−2 × Ic)> 0.8), T / T homozygous , (Ia−Ic) / (Ia + It−2 × Ic)> 0.8 (or (It−Ic) / (Ia + It−2 × Ic) <0.2), C / C homozygous; When 4 <(Ia−Ic) / (Ia + It−2 × Ic) <0.6 (or 0.4 <(It−Ic) / (Ia + It−2 × Ic) <0.6), C / T It was decided to be heterozygous.
[0073]
4. result
FIG. 6 shows the results of analyzing 325 genomic samples for SNP of NCBI AF 538844. The horizontal axis in FIG. 6 represents (Ia−Ic) / (Ia + It−2 × Ic), and the vertical axis represents frequency. A group consisting of 81 alleles derived from a homozygote of C / C, 83 homozygotes of T / T, and 82 heterozygotes of C / T was formed. As a result of analyzing the same SN site of the same sample by DNA sequencing, it was found that the results of all the samples were consistent with the results of this example. This SNP analysis using biochemiluminescence is performed by extremely simple operations such as 1) addition of a reagent for decomposing impurities to a genomic sample, 2) PCR amplification, and 3) addition of a luminescence reagent to a PCR product. There is an advantage that it can be done.
[0074]
【The invention's effect】
According to the present invention, the sequence of a specific site on a target nucleic acid can be determined accurately and in a short time by performing PCR amplification with a set of primers whose 3 ′ end corresponds to a specific site (eg, an SNP site). Can be. If a genome sample has been prepared, the determination can be made within 30 minutes. In particular, a system using biochemiluminescence does not require expensive and complicated optical equipment and can be measured with a normal simple luminometer, so that miniaturization and automation of the device system can be expected. Furthermore, in this system, by preparing primers corresponding to each SNP, it is possible to determine a plurality of SNPs derived from a plurality of genomic samples, and there is an advantage that the system is extremely versatile.
[0075]
[Sequence list]

[0076]
[Sequence List Free Text]
SEQ ID NO: 1—Description of Artificial Sequence: Sense Primer
SEQ ID NO: 2—Description of Artificial Sequence: Antisense Primer
SEQ ID NO: 3—Description of Artificial Sequence: Sense Primer
SEQ ID NO: 4—Description of Artificial Sequence: Antisense Primer
SEQ ID NO: 5-Description of artificial sequence: PCR amplification product
SEQ ID NO: 6-Description of artificial sequence: PCR amplification product
[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of an SNP analysis method by a BAMPER method.
FIG. 2 is a diagram showing the principle of the SNP analysis method according to the present invention.
FIG. 3 is a graph showing the results of electrophoresis in SNP analysis of p53 exon 8 according to the present invention.
FIG. 4 is a graph showing the results of electrophoresis when PCR is performed at different denaturation temperatures (80 ° C. and 94 ° C.) in the SNP analysis of p53 exon 8.
FIG. 5 is a graph showing PCR temperature characteristics of the present invention. A is a graph showing the relationship between the denaturation temperature and the relative amplification amount, and B is a graph showing the relationship between the base length and the dissociation temperature.
FIG. 6 is a graph showing the results of analysis of a large amount of a sample using the present invention.
[Explanation of symbols]
1 ... sense strand of PCR product, 1 '... antisense strand of PCR product
2. dNTPs and primer residue
3: SNP part in the sense strand
4: SNP part in antisense strand
5… SNP inspection probe
6 ... Extended chain
7 ... pyrophosphoric acid
21: Genome sense strand, 21 ': Genome antisense strand
22: SNP portion in genome sense strand, 22 ': SNP portion in genome antisense strand
23… SNP probe that binds to genome sense
24 ... SNP probe that binds to genomic antisense
25, 26 ... identification sequence corresponding to SNP
27, 28: Sequence part that is not complementary to the genome sequence
29, 30 ... hybrid of each strand of genome and probe for SNP judgment
31 ... PCR product
41 ... A homozygote tote specimen
42 ... T homozygote tote specimen
43 ... Heterozygote sample
44, 45, 46 ... Electrophoresis results with SNP test probe with T at the end
47, 48, 49: Electrophoresis results with SNP test probe whose end is A
50, 51, 52, 53 ... peak of PCR product
54: Probe peak
61: PCR result at 80 ° C denaturation temperature
62: PCR results at a denaturation temperature of 94 ° C
63… Target PCR product
64: Probe
65: False positive amplification peak
71: Relative value of electrophoretic separation peak area derived from target PCR product
72: Relative value of the sum of peak areas related to false positive amplification products
81: Allele A sample group
82: Heterozygote sample group
83 ... Allele T sample group

Claims (10)

A nucleic acid amplification method for obtaining an amplification product in a desired base length range including a specific site on a target nucleic acid,
A first primer comprising a sequence complementary to the base sequence of the first strand of the target nucleic acid and having a 3 ′ end designed to hybridize to the specific site on the first strand; and a second primer of the target nucleic acid. Performing nucleic acid amplification using a second primer containing a sequence complementary to the base sequence of the strand and having a 3 ′ end designed to hybridize to a site forming a base pair with the specific site on the second strand. And controlling the denaturation temperature in the nucleic acid amplification to dissociate the amplification product in the desired base length range into a single strand and use it as a template for the next nucleic acid synthesis reaction. Method. The nucleic acid amplification method according to claim 1, wherein the denaturation temperature in the amplification step is controlled to 60 to 85C from at least the fifth cycle. The amplification product having the desired base length range is a nucleic acid synthesized by hybridizing a 3 ′ end of the first primer and the second primer to a specific site on the target nucleic acid. 3. The nucleic acid amplification method according to item 1. The nucleic acid amplification method according to any one of claims 1 to 3, wherein each of the first primer and the second primer has a base length of 18 to 50. The nucleic acid amplification method according to any one of claims 1 to 4, wherein the melting temperature of the first primer and the melting temperature of the second primer are substantially the same. The nucleic acid amplification method according to any one of claims 1 to 5, wherein the sum of the ratios of guanine and cytosine in the first primer and the second primer is 35 to 65%. A method for analyzing a sequence of a specific site on a target nucleic acid,
A first primer comprising a sequence complementary to the base sequence of the first strand of the target nucleic acid and having a 3 ′ end designed to hybridize to the specific site on the first strand; and a second primer of the target nucleic acid. Performing nucleic acid amplification using a second primer containing a sequence complementary to the base sequence of the strand and having a 3 ′ end designed to hybridize to a site forming a base pair with the specific site on the second strand. And controlling the heating temperature in the nucleic acid amplification to dissociate the amplification product in the desired base length range into a single strand, and to use it as a template for the next nucleic acid synthesis reaction, And the step of analyzing the sequence of a specific site on the target nucleic acid by detecting the amplification product. The method according to claim 7, wherein the specific site is a single nucleotide polymorphism site. The method according to claim 7, wherein the detection of the amplification product is performed by a biochemiluminescence reaction using pyrophosphate generated in accordance with nucleic acid amplification. A system for analyzing the sequence of a specific site on a target nucleic acid,
A reaction vessel containing a target nucleic acid and a reaction solution containing primers for obtaining an amplification product in a desired base length range including a specific site on the target nucleic acid,
A temperature control mechanism for controlling the temperature in the reaction vessel to dissociate and amplify the amplification product in the desired base length range,
Having a light detection mechanism for detecting light emission generated in the reaction tank, and
The system according to claim 1, wherein the system recognizes a type of the primer that has generated the biochemiluminescence and analyzes a sequence of a specific site contained in the target nucleic acid.

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