JP3974557B2 - Mutation analysis method and system therefor - Google Patents

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]
BACKGROUND 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 group, and a system for the method.
[0002]
[Prior art]
One important issue of “post-genome research”, the next research after human genome decoding, is single nucleotide polymorphism analysis to examine minute differences in sequences among individuals. The sum of single nucleotide polymorphisms (SNPs) in humans means the individual “individuality” itself. SNP appears at a frequency of 1,500 bases in the human genome, and it is thought that there are many that affect susceptibility to disease, ease of healing, sensitivity to drugs, or environmental stress response. Yes. Therefore, as post-genomic research progresses, the difference in the mechanisms that cause the underlying disease, even for diseases with the same diagnostic name and similar symptoms, will become clear at the molecular level. And it is expected that medical individualization (made-to-order) such as proper use of drugs taking into account the difference will be possible. In addition, it becomes possible to determine the risk of individual susceptibility to disease (susceptibility to disease), and by building a lifestyle to avoid the disease as necessary, disease prevention, onset delay, early detection・ Early treatment can be realized.
[0003]
SNPs are gene sequences in the meiotic genome that have been replaced with other bases, which are inherited genetically by mating. Therefore, when the sequence of a group consisting of many individuals is examined, two types of sequences at a specific site of the gene appear at a certain frequency. SNPs are classified into five types according to their location. SSNP that exists in the translation region without amino acid sequence change, cSNP with amino acid change or protein cannot be made, rSNP or iSNP that exists in the regulatory region and may affect gene expression level, and other regions However, gSNP has almost no effect on gene expression level.
[0004]
All SNPs are useful as polymorphic markers for finding genes related to the onset and exacerbation of diseases, but important in the clinical field are cSNPs and rSNPs that are directly related to disease risk diagnosis and proper use of drugs. Estimate the number of SNPs considered medically important. There are 3 to 6 million SNPs in the human genome consisting of 3 billion base pairs. As a result of the Human Genome Project, it is known that the number of human genes is approximately 26,000-28,000 and the average size is 1 kb (1,000 bases). For this reason, the area | region where meaningful SNP exists is narrowed down to the range of 26-28 Mb. In protein translation from a gene, amino acids are determined by a triplet codon, which is a sequence combination of 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 with similar properties, so that only 2/3 of the total affects the function of the protein. Therefore, SNPs involved in protein function are present in the range of approximately 17-19 Mb. If the frequency of occurrence of SNP is one in 1.5 kb, the maximum number of SNPs involved in the function is 13,000. One of the factors that determines the SNP analysis market in the medical field is how much of an individual SNP is meaningful for analysis. In other words, even if cSNP and rSNP are combined, there is a high possibility that useful SNPs will be narrowed down to thousands of the same number as 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. The search requires a method for mass analysis of SNPs, and many methods and devices have been reported for this purpose. For example, as a method for searching for SNP candidates from completely unknown regions, there is a conventional DNA sequencing method in which sequences of specific regions are analyzed and compared with a plurality of samples. In addition, SNP methods that have been narrowed down to a certain extent include SSCP methods that utilize different stabilization energy when DNA forms a higher-order structure, and denaturant concentration gradient gel electrophoresis. (DGGE), heat-denaturing HPLC method (WAVE (Registered trademark) system), a method for selectively chemically cleaving a chimeric double-stranded SNP site obtained by mixing a sample containing an SNP site (CCM), an S1 nuclease method for enzymatically cleaving the chimeric portion, Invader method for typing in SNP, TaqMan method, DNA chip method for comparing differences in hybridization stability with probe, mini-sequence method for determining local sequence of only SNP base site (for example, see Non-patent Document 1) A mass array method combining mini-sequence and mass spectrometry has been developed. Furthermore, as an analysis method for the purpose of clinical examination 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 carried out using two types of probes designed so that the 3 'end of the target gene matches the SNP allele, and the pyrophosphate produced at that time is converted into a luciferin-luciferase reaction. Detected by biochemiluminescence. Since the extension reaction proceeds only when the mutation corresponding to the probe used is present in the target gene, the SNP sequence of the target gene can be determined within a few minutes by measuring luminescence. The BAMPER method is considered to be an analysis method suitable for clinical use in that it can perform SNP analysis simply by adding a reagent and can be performed with a simple optical luminescence measuring device.
[0007]
However, SNP analysis by the BAMPER method requires a four-step reaction 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:
The purpose of this step is not only to amplify the sample, but also to amplify a limited region including the SNP site, thereby reducing the subsequent false positive amplification reaction of the 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 and the dNTP and primer which is the reaction residue 2 is formed.
[0008]
(2) PCR product purification from PCR reaction solution:
In the BAMPER reaction, pyrophosphate generated in the probe extension reaction is detected, so PCR residue 2 consisting of primers and dNTP used in PCR interferes with the measurement. That is, the PCR primer hybridizes to the PCR product and causes complementary strand synthesis by DNA polymerase to produce pyrophosphate. DNTP also serves as a pseudo-substrate for the luminescence reaction for detecting pyrophosphate. Therefore, in order to remove these, in FIG. 1, 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 is performed with a biotin-labeled primer, and excess PCR primers and dNTP are removed by capturing the amplified product on magnetic beads with streptavidin. . The supplemented PCR product can be alkali-denatured so that only the strand immobilized on the beads can be used as a template for the next BAMPER extension reaction.
[0009]
(3) First stage of BAMPER reaction (complementary chain extension reaction with BAMPER probe):
The purified template PCR product is hybridized with a 20-30 base long BAMPER probe having a structure in which the 3 'end of probe 5 is complementary to the SNP site, and a complementary strand extension reaction is performed by the catalytic action of DNA polymerase. Make it. Here, in order to increase the fidelity of the probe extension reaction corresponding to the SNP sequence, a base sequence that mismatches with the sample DNA sequence is inserted in advance at the 3rd to 4th bases from the 3 ′ end of the probe. (For example, refer nonpatent literature 2 and 3.).
[0010]
Only when the probe has a sequence complementary to the SNP site at the 3 'end, an extension reaction occurs, producing 6 extended strands and 7 pyrophosphate. Since one molecule of pyrophosphate is generated per base extension, the amount of pyrophosphate increases with the extension reaction. Probes that are non-complementary at the 3 'end do not undergo an extension reaction and do not produce pyrophosphate. In order to increase the amount of pyrophosphoric acid produced in the normal reaction cycle, the denaturation step at 94 ° C. (10 seconds) and the hybridization and extension reaction step (10 seconds) at 50-60 ° C. are repeated about 5 times. Since the DNA extension chain length in the BAMPER reaction is about 200 bases at the longest, the extension reaction occurs sufficiently even in a short reaction cycle.
[0011]
(4) Second 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 with luciferin-luciferase. That is, luminescence around 530 nm, which is generated when the excited oxidized luciferin 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 caused light emission.
[0012]
[Patent Document 1]
JP 2002-101899 A
[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, the SNP analysis by the BAMPER method requires a four-step reaction including two complementary strand synthesis reactions. If this can be carried out with fewer steps, the operation is further simplified, and it is expected that the processing will be automated.
[0014]
That is, an object of the present invention is to improve the SNP analysis technique based on the BAMPER method, and to provide a simple and inexpensive SNP analysis method and a system therefor that are more suitable for clinical use.
[0015]
[Means for Solving the Problems]
As a result of diligent investigations to solve the above problems, the inventors of the present invention, in the SNP analysis by the BAMPER method, both the PCR amplification (1) and the complementary strand extension reaction by the BAMPER probe (3) are complementary to each other by DNA polymerase. We focused on the chain synthesis reaction. Then, if either primer used for PCR was designed so that the 3 'end coincides with the SNP site in the same manner as the BAMPER probe, it was thought that complementary strand extension reaction by PCR and BAMPER probe could be performed together.
[0016]
However, since false positive amplification is actually caused by non-specific hybridization of primers, it is difficult to specifically detect the SNP sequence only by the above method. Therefore, the inventors have further studied and devised primers and PCR conditions to selectively amplify a short region near the SNP and detect the SNP sequence in 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 having 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 designed to vibrate the 3 ′ end at the specific site on the first strand; and a second primer of the target nucleic acid Nucleic acid amplification is performed using a second primer that contains a sequence complementary to the base sequence of the strand and is designed to vibrate the 3 ′ end at a site that makes a base pair with the specific site on the second strand. ,as well as
By controlling the denaturation temperature in the nucleic acid amplification, the amplification product in the desired base length range is dissociated into single strands and used 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 (for example, around 94 ° C.) to advance the amplification reaction, and when the short-chain amplification product from the primer increases to some extent, it is lowered to 60-85 ° C. To control. Specifically, it is preferable to control to lower 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 ′ ends of the first primer and the second primer to a specific site on the target nucleic acid. Therefore, the desired base length range is a value defined by the base length 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]
It is desirable that the first primer and the second primer have a base length of 18 to 50 bases and have substantially the same melting temperature. The sum of the ratios of guanine and cytosine in the first primer and the second primer is preferably 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 a first primer comprising a sequence complementary to the base sequence of the first strand of the target nucleic acid and designed to vibrate the 3 ′ end at the specific site on the first strand, and the target 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 vibrate at a site which makes a base pair with the specific site on the second strand Performing amplification, and
A nucleic acid amplification step characterized by controlling the heating temperature in the nucleic acid amplification to dissociate the amplification product in the desired base length range into single strands and use as a template for the next nucleic acid synthesis reaction; and
A step of analyzing a sequence of a specific site on the target nucleic acid by detecting the amplification product.
[0022]
Here, examples of the specific site to be detected include a single nucleotide polymorphism site.
In addition, as a method for detecting an amplification product, any known method such as electrophoresis may be used. However, a method for detecting pyrophosphoric acid produced in response to nucleic acid amplification by a biochemiluminescence reaction requires a device and an operation. It is preferable in terms of simplicity.
[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 having 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;
A light detection mechanism for detecting luminescence generated in the reaction vessel, and
Recognizing the type of primer that produced the biochemiluminescence, the sequence of a specific site contained in the target nucleic acid is automatically analyzed.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
In the SNP analysis method of the present invention, the three steps of (1) PCR amplification, (2) purification, and (3) SNP-specific complementary chain extension reaction required in the BAMPER method are devised by devising primers and PCR conditions. The process is performed by a single operation, and the operation is simplified and speeded up.
[0025]
In particular, the present invention is characterized by overcoming the problem of false positive amplification, which is a problem during nucleic acid detection, by devising the above primers and PCR conditions. Hereinafter, the present invention will be described in detail.
[0026]
1 Primer
1) Primer function
In the present invention, a short region in the vicinity of a specific site is selectively amplified by using a primer set designed so that all of its 3 'ends coincide with a specific site to be detected (for example, an SNP site). 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 3 'end of both the primer corresponding to the sense strand and the antisense strand so as to coincide with the SNP site. That is, even if a false positive amplification in which the primer is hybridized non-specifically to the genome occurs, the minimum product in the PCR product is the target true amplification product. Therefore, by controlling the denaturation temperature during PCR amplification described later, false positive amplification can be prevented by selectively dissociating a 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 first primer containing a sequence complementary to the base sequence of the first strand of the target nucleic acid and designed to vibrate the 3 ′ end at a specific site on the first strand, and the first primer of the target nucleic acid. A pair of primers comprising a second primer designed to vibrate the 3 ′ end at a site that forms a base pair with the specific site on the second strand, the sequence comprising a sequence complementary to a double-stranded base sequence Composed. In the case of single nucleotide polymorphism detection, this primer set requires at least 2 sets corresponding to each allele, and in some cases 3 to 4 sets. That is, as the sequence at the 3 ′ end of the primer, A / C group, A / G group, A / T group, C / G group, C / T group, G / T group, A / C / G group, A Primer pairs of / C / T, A / G / T, C / G / T, and A / C / G / T are considered.
[0029]
3) Introduction of mismatch
The above primer introduces a structure (mismatch) different from the sequence of the corresponding genomic sample at the position of the 3rd to 4th bases on the 5 'side from the 3' end of at least one of the primers, if possible. It is preferable to do. Thereby, the mutation specificity (for example, SNP allele) sequence specificity of the amplification reaction in PCR is remarkably increased. In the conventional BAMPER method, the same mismatch is introduced into the primer, but in normal PCR, the primer is designed to hybridize to a position away from each other, so that only one primer is artificially non-complementary. The site (mismatch) cannot be introduced. Since the primer of the present invention has a structure in which the 3 'ends overlap, it is possible to make mismatches in both primers. Thereby, the accuracy of analysis is remarkably improved.
[0030]
In PCR using a primer set in which the 3 'end coincides with the SNP position, there is no degree of freedom in designing the PCR primer, and the sequence position is determined almost uniquely. For this reason, when a palindrome appears in a primer, the primer itself has a higher-order structure, and the case where hybridization with a template DNA is inhibited frequently occurs. In order to avoid this, when at least one of the primers has a structure that forms a palindrome such as 4 bases, 6 bases, or 8 bases, any one of the half from the 5 ′ end side of the sequence site that forms the palindrome The palindromic structure is eliminated by making one base different from the original sequence.
[0031]
4) Base length
The primer used in the present invention is preferably 18 to 50 bases in length, particularly about 18 to 30 bases in the same manner as ordinary PCR primers.
In the present invention, 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 ′ ends of the first primer and the second primer to a specific site on the target nucleic acid is the first primer and the second primer. Defined by the base length of the primer. That is, the base length of the true amplification product is “a 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, the desired base length range is 35 to 99 bases.
[0032]
5) Melting temperature (Tm)
In the present invention, it is preferable that the melting temperatures (Tm) of the first primer and the second primer are substantially the same. Here, “substantially the same” means that the calculation is not completely the same, but the difference is as small as 5 ° C or less and can be treated as almost the same Tm in the experiment. To do.
[0033]
6) GC content (%)
The sum of the ratios of guanine and cytosine (GC content (%)) of 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 approximately equal to the sum of the GC contents (%) of both primers. The Tm of nucleic acid varies depending on its base length and GC content (%), but as will be described later, short chains (35-99) with a length of 35-99 bases at a denaturation temperature of 60-85 ° 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 desirably 35% to 65%, particularly 40% to 60%. Therefore, the sum of the GC contents (%) of both primers is desirably 35% to 65%, particularly 40% to 60%.
[0034]
2. PCR conditions
Another feature of the present invention is temperature control during PCR amplification. In the nucleic acid amplification step, it is necessary to change the temperature sequentially to the denaturation temperature for dissociating the template nucleic acid into a single strand, the temperature for annealing, and the temperature for extension reaction, as in the normal PCR reaction cycle. is there. Particularly in the case of the present invention, the denaturation temperature in nucleic acid amplification is initially high as usual, for example, 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 at 60 to 85 ° C., preferably 80 to 85 ° C. from at least the fifth cycle. Thus, only a short true amplification product can be dissociated, and even if a false positive amplification occurs, the product does not become a template for the next nucleic acid synthesis.
[0035]
Of course, the denaturation temperature of the PCR product varies depending on the base length and GC content (%) of the extension product. For example, if the GC content (%) is 58%, the PCR product is 60 bases long, the Tm is 71 ° C, 80 bases long is 76 ° C, 100 bases long is 77 ° C, 150 bases long is 79 ° C, 200 bases long 81 ° C. (see FIG. 5B). In practice, the denaturation temperature of PCR needs to be 5 to 10 ° C. higher than these Tm values. Therefore, when the base length of the PCR product is 80 bases, the denaturation temperature is preferably 80 to 85 ° C. (see FIG. 5B). In other words, when the GC content (%) is 58%, a PCR product that can be amplified at a denaturation temperature of 80 ° C. has a limit of 80 bases.
[0036]
On the other hand, when 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 a denaturation temperature of 80 ° C. becomes longer). Further, when the GC content (%) increases, the Tm value also increases, and the preferable denaturation temperature also rises (the base length of the PCR product that can be amplified at a denaturation temperature of 80 ° C. becomes shorter).
[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. However, such a short amplification product has a GC content in the range of 35 to 65% and 60 to 60 bases. Denaturation is possible in the range of 85 ° C.
In the present invention, in order to adjust the Tm of the amplification product, a Tm adjusting agent or the like may be used if necessary.
[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 of the specific site can be determined by the sequence of the 3 'end of the primer set that generated the amplification product.
[0039]
In the present invention, detection of a desired true amplification product may be performed using any known detection method such as electrophoresis or fluorescence detection using energy transfer, but detection using biochemiluminescence described below. The method is preferred in that the apparatus 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 an extension reaction of one base. Therefore, by detecting this pyrophosphate, it becomes possible to detect the amplification product indirectly.
[0041]
The pyrophosphate is detected using biochemiluminescence by luciferin-luciferase, as in the BAMPER method. That is, it is first converted to ATP using pyrophosphate diphosphokinase or ATP sulfurylase. When the converted ATP and luciferin are oxidatively reacted using luciferase as an enzyme, oxyluciferin, pyrophosphate, and a series of other substances are produced. Light emission around 530 nm, which is generated when the generated excited state luciferin returns to the ground state, is detected. Since light emission can generate only a primer complementary to the sequence of a specific site (for example, SNP site) on the target nucleic acid, the sequence of the specific site can be determined based on the sequence of the 3 'end of the primer that generated the light emission.
[0042]
In biochemiluminescence using luciferin-luciferase, ATP or dATP, which is a substrate for pyrophosphate or luciferase, must not be included in the sample, and short chains that cause non-specific complementary strand synthesis reactions. There must be no DNA or single-stranded parts. Therefore, pyrophosphate, ATP, and dATP contained in the sample are previously degraded by phosphatase, and short-chain DNA and single-stranded portion are degraded by exonuclease I.
[0043]
4). Mutation analysis system
When the method of the present invention is carried out using biochemiluminescence, separation / analysis means such as electrophoresis and chromatography are unnecessary, and excitation by a laser or the like is not necessary, so that it is suitable for automation of operation. . The present invention provides a system for such automated analysis.
[0044]
For example, the system controls a temperature of the reaction vessel containing a reaction solution containing a target nucleic acid and a primer for obtaining an amplification product having a desired base length range including a specific site on the target nucleic acid, and 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 the biochemiluminescence was generated. The sequence of a specific site included in the target nucleic acid is automatically analyzed by recognizing the kind of primer.
[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 the template nucleic acid into single strands, annealing temperature). In addition to the temperature for the extension reaction and the temperature for the extension reaction, the denaturation temperature in the nucleic acid amplification is initially set as high as usual (for example, around 94 ° C.) This means a control mechanism that lowers the denaturation temperature to 60-85 ° C. when the short-chain true amplification product from the nuclei increases to some extent (for example, after the fifth cycle).
The above system can be suitably used for mutation analysis such as SNP analysis in a clinical site, for example.
[0046]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, 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 SNP, amplified using the method of the present invention, and analyzed by electrophoresis.
[0048]
1. Apparatus and sample
As the thermal cycler, DNA Engine PTC200 (MJ Research) was used. A microchip electrophoresis analysis system SV1210 (Hitachi Electronics Engineering) was used for confirmation of PCR products. Wallac 1420 ARVOsx (PerkinElmer) was used as a luminometer. Primer synthesis was outsourced to SIGMA.
[0049]
As a sample, a human-derived genome was used. The genome was hemolyzed by adding 9 volumes 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. To this precipitate, 1 ml of DNAzol was added to dissolve 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 ethanol precipitation. Finally, it was dissolved in sterilized water so that the genome concentration was 2.5 to 25 ng / μl to obtain a sample genome solution. The prepared sample genome was previously identified for the SNP allele, and two types of homozygotes (AA), (TT), and heterozygotes (AT) were used for the following experiments, respectively.
[0050]
2. Method
This embodiment will be 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 complementary strand) is the target displacement site. This part is not exactly SNP but merely a mutated part of the sequence, but 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 '. In the present invention, the primers 23 and 24 are used.
[0051]
The two primers used are: primer 24 is substantially complementary to double-stranded antisense strand 21 'of the genomic sample and the other primer 23 is substantially to sense strand 21 which is the other strand of the genome. Complementary to The 3 'ends 25 and 26 of both primers are complementary to the bases of the SNP alleles 22 and 22' to be detected, respectively. This time, the SNP to be detected is the part corresponding to Cys275Ser present in p53 exon 8. This is a polymorphism of the 275th codon as the amino acid sequence, and the allele of the base sequence is A / T. Therefore, two primer sets (23 and 24) were prepared, one for detecting an allele whose SNP site is A, and one for detecting an allele whose SNP site is T.
[0052]
Furthermore, sequences 27 and 28 that are non-complementary to p53 exon 8 (sample DNA) were introduced into the third base from the 3 'end of each primer in order to enhance selectivity. That is, the third base from the 3 'end of the primer complementary to the antisense strand was altered from T to A corresponding to the original sequence of p53 exon 8. The primer complementary to the sense strand was modified 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, 2 out of the 3 bases near the 3' end of the primer are completely complementary and DNA polymerase causes complementary strand extension, but the 3 'end is not complementary to the allele. In such a primer set, the 3 ′ end and the 2nd base of the third base are non-complementary, so that the vicinity of the primer 3 ′ end cannot hybridize to the template DNA, and complementary strand extension by DNA polymerase hardly occurs.
[0053]
Primer 23 was 20 bases long and primer 24 was 22 bases long and was designed so that the Tm during 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 optimal temperature during PCR hybridization (during annealing) was 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 alleles with SNP site A
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 alleles with SNP sites
sense primer: 5'-aac agc ttt gag gtg cgt gAt A-3 '(SEQ ID NO: 3, 22 base length) antisense primer: 5'-tct ctc cca gga cag gcT cT-3' (SEQ ID NO: 4, 20 base length) ).
[0055]
The PCR product obtained by the above primer has a length of 41 bases (bp), and the one corresponding to allele A is 5'-aac agc ttt 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). These Tm are calculated to be 65.4 ° C.
[0056]
2 μl of genomic DNA sample prepared to 2.5 to 25 ng / μl was added to a 96-well PCR plate and placed on ice. 5 unit / μl Taq.DNA polymerase (QIAGEN) 0.05 μl, 2.5 mM dNTPs 1 μl, 25 μmol / μl of primer pairs corresponding to each allele (use sterile water for control), mix 1 μl Further, sterilized water and PCR buffer were added to prepare a final solution of 23 μl per well. In practice, it is more efficient to make one reaction sample containing Taq.DNA polymerase, dNTPs, and primer set more than the required amount in advance, and add 23 μl each to each well, and PCR reproducibility is also higher. . In addition, about each genomic DNA sample, what added sterilized water instead of the primer group as a control was used for reaction similarly.
[0057]
Next, the upper part of the PCR plate was sealed with an adhesive sheet, and 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 PCR performed using two sets of primers corresponding to the SNP sequence and analysis by electrophoresis 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, 44, 45 and 46 are electrophoretic separation patterns when the PCR is performed with a primer set whose allele is complementary to A, and 47, 48 and 49 are primer sets whose allele is complementary to T.
[0059]
From the homozygous (A / A) genome, a specific amplification peak 50 was obtained only when amplified with a primer set complementary to A. In addition, from the homozygous (T / T) genome, a specific amplification peak 51 was obtained only when amplified with a primer set complementary to T. On the other hand, specific amplification peaks 52 and 53 were obtained from the heterozygote (A / T) genome when amplified with either a primer set complementary to A or a primer set complementary to T. The peak 54 detected in common in all the electrophoresis patterns corresponds to the probe.
[0060]
In this PCR system, the amplified base length is as short as 41 bases, so the temperature cycle may be as short as 50 ° C. for 10 seconds and 80 ° C. for 10 seconds. In addition, since the denaturation temperature is as low as 80 ° C., there is an advantage that less time is required for the temperature to rise and fall. In fact, the time required for PCR is calculated with a temperature rise and fall rate of 3 ° C / second, increasing from 55 ° C to 80 ° C per cycle for about 10 seconds, 80 ° C for 10 seconds, and falling from 80 ° C to 55 ° C. It takes about 40 seconds for 10 seconds at 55 ° C. In other words, the entire process will be completed in about 20 minutes. In the case of this example, since the time required for microchip electrophoresis is about 3 minutes, it was confirmed that SNP determination (typing) from the genome can be performed in a short time even from PCR to detection.
[0061]
Example 2: Examination of PCR conditions
Using primers and genomes corresponding to the alleles shown in Example 1, the denaturation temperature dependence of the false positive amplification reaction was examined.
[0062]
FIG. 4 shows an electrophoretic separation pattern of amplification products when the higher temperature in the PCR cycle is 94 ° C., which is the same as in normal PCR, and 80 ° C. used in the present invention. 63 is the target PCR amplification product. 64 is a peak derived from a probe. A series of peaks 65 appearing in the pattern 61 are dependent on quasi-positive amplification. That is, it was confirmed that false positive amplification can be prevented by lowering the higher temperature of 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, experiments were conducted in the same manner at 75 ° C., 80 ° C., 85 ° C., 90 ° C., 95 ° C., and 97 ° C. at the denaturation temperature of PCR (the higher temperature), and the comparison results are shown in FIG. 5A. . In the figure, 71 plots indicated by ● represent relative values of the area of the electrophoresis separation peak 63 derived from the target PCR product. The 72 plots indicated by Δ in the figure represent the relative values of the peak areas related to the false positive amplification product 65 observed in FIG. Here, the relative value of the area is calculated by setting the area value of the temperature when the peak area is maximum in the peak series of 61 and 62 as 1.0.
[0064]
FIG. 5A shows that the target PCR product is obtained when the denaturation temperature is in the range of 80 to 85 ° C., but the false positive amplification is below the detection limit. On the other hand, at 90 ° C. or higher, the target product can be obtained, but it can be seen that false positive amplification increases. The amount of product decreased at 97 ° C. because the enzyme was heat denatured.
[0065]
Of course, the denaturation 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 PCR product has a base length of 41 bases and a calculated Tm = 65 ° C. If the GC content (%) is 58%, the Tm is 71 ° C when the PCR product is 60 bases long, 76 ° C for 80 bases long, 77 ° C for 100 bases long, 79 ° C for 150 bases long, 81 ° C for 200 bases long It becomes ℃. In practice, the denaturation temperature of PCR needs to be 5 to 10 ° C. higher 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, PCR products that can be amplified at a denaturation temperature of 80 ° C. have a limit of 80 bases in length.
[0066]
Example 3: Mutation analysis of p53 exon 8 using biochemiluminescence
Using the sample genome and primer set prepared in Example 1, the mutant portion corresponding to Cys275Ser present in p53 exon 8 was analyzed using biochemiluminescence by 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 and luciferase, the sample must not contain ATP or dATP, which is a substrate for pyrophosphate or luciferase, and short DNA that causes nonspecific complementary strand synthesis reactions There must be no single-stranded parts. Therefore, in this example, it is necessary to decompose pyrophosphate, ATP, and dATP contained in the genome prepared by the method of Example 1 in advance with phosphatase, and to decompose short-chain DNA and single-stranded part with exonuclease I. is there.
[0068]
That is, in a well of a 384-well PCR plate, 2 μl of genomic DNA sample prepared at 8-56 ng / μl, 1 μl of shrimp alkaline phosphatase (SAP) at a concentration of 1 unit / μl, 10 unit / μl of exonuclease I A mixture of 0.1 μl of (ExoI), 0.5 μl of 10 × PCR buffer (Amersham Pharmacia) and 1.4 μl of sterilized water was added. The plate is spun down to collect liquid on the wall. The liquid 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 placed on top of the weight to seal and heat-insulate. 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, the temperature was raised to 95 ° C for 5 seconds to denature the genome, and then rapidly cooled on ice. .
[0070]
1 μl of the genomic sample solution after the above pretreatment was dispensed into three wells of the PCR plate. 5 unit / μl Taq.DNA polymerase (QIAGEN product) 0.02 μl, 2.5 mM dNTPs 0.4 μl, 25 pmol / μl of each primer set corresponding to each 0.08 μl (control uses sterile water), 10 × PCR buffer (Amersham Pharmacia, containing 15 mM Mg ions) 0.8 ml, 10 mM MgCl₂0.45 ml (final magnesium concentration at the time of PCR is 2 mM) and sterilized water were added so that the solution per well became 9 μl. All operations so far were performed on ice. In addition, about each genomic DNA sample, what added sterilized water instead of the primer group as a control was used for reaction similarly.
Each well was overlaid with 5 μl of mineral oil to prevent drying, and then subjected to a PCR cycle reaction. In PCR, a cycle of 55 ° C. for 10 seconds and 80 ° C. for 10 seconds was repeated 35 times in total.
[0071]
3. Detection by biochemiluminescence
In Example 1, PCR products were separated and analyzed by electrophoresis. In this example, PCR products were detected using biochemiluminescence. 18 μl of a luminescent reagent (Kikkoman Co., Ltd.) that had been returned to room temperature in advance was added to the PCR-completed sample consisting of 3 wells as a set, pipetted 5 times, mixed, 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. This reagent has the advantage that the reagent is stable and quality control is easy because thermostable recombinant Japanese-produced Genji firefly-derived luciferase is used.
[0072]
Allele judgment is derived from allele C, where Ia is the signal value obtained from the primer set complementary to allele C, It is the signal value obtained from the primer set complementary to allele T, and Ic is the signal value from the control without primer. The signal intensity ratio (Ia-Ic) / (Ia + It-2 × Ic) and the signal intensity ratio derived from allele T (It-Ic) / (Ia + It-2 × Ic) were calculated. Judgment criteria are T / T homozygous when (Ia-Ic) / (Ia + It-2 × Ic) <0.2 (or (It-Ic) / (Ia + It-2 × Ic)> 0.8) , (Ia-Ic) / (Ia + It-2 × Ic)> 0.8 (or (It-Ic) / (Ia + It-2 × Ic) <0.2), C / C homozygote, 0.4 < When (Ia-Ic) / (Ia + It-2 × Ic) <0.6 (or 0.4 <(It-Ic) / (Ia + It-2 × Ic) <0.6), it is judged as a heterozygote of C / T It was decided to.
[0073]
4). result
FIG. 6 shows the result of analyzing SNP of NCBI AF 538844 for 325 genomic samples. The horizontal axis of FIG. 6 represents (Ia−Ic) / (Ia + It−2 × Ic), and the vertical axis represents frequency. A group of alleles derived from C / C homozygotes was 81, T / T homozygotes were 83, and C / T heterozygotes were 82. As a result of analyzing the same SN site of the same sample by DNA sequencing, it was found that all the samples matched the result of this example. This SNP analysis using biochemiluminescence enables SNP determination with extremely simple operations such as 1) addition of reagents that decompose impurities into genomic samples, 2) PCR amplification, and 3) addition of luminescent reagents to PCR products. There is an advantage that 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, SNP site). Can do. If a genomic sample is prepared, the determination can be made within 30 minutes. In particular, a system using biochemiluminescence does not require expensive and complicated optical system equipment, and can be measured with a normal simple luminometer, so that downsizing and automation of the apparatus system can be expected. Furthermore, in this system, by preparing a primer corresponding to each SNP, it is possible to determine a plurality of SNPs derived from a plurality of genome samples, and there is an advantage that it is extremely versatile.
[0075]
[Sequence Listing]

[0076]
[Sequence Listing 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 was performed at different denaturation temperatures (80 ° C. and 94 ° C.) in the SNP analysis of p53 exon 8.
FIG. 5 is a graph showing the 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 a large amount of specimen analysis using the present invention.
[Explanation of symbols]
1 ... sense strand of PCR product, 1 '... antisense strand of PCR product
2… dNTP and primer residue
3 ... SNP part in the sense strand
4… SNP part in the antisense strand
5… SNP inspection probe
6 ... Elongation chain
7… Pyrophosphate
21 ... sense strand of genome, 21 '... antisense strand of genome
22 ... SNP part in genome sense strand, 22 '... SNP part in genome antisense strand
23… SNP probe binding to genome sense
24… SNP probe binding to genomic antisense
25, 26 ... Identification sequence corresponding to SNP
27, 28 ... Sequence parts that are non-complementary to the genome sequence
29, 30 ... Hybrid of each strand of genome and probe for SNP determination
31 ... PCR products
41 ... A homozygous tote sample
42 ... T homozygote sample
43… Heterozygote specimen
44, 45, 46 ... Electrophoresis result with SNP probe with T terminal
47, 48, 49 ... Electrophoresis results with SNP probe with A terminal
50, 51, 52, 53 ... Peak of PCR product
54… Peak peak
61… PCR results at a denaturation temperature of 80 ° C
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 electrophoresis separation peak area derived from target PCR product
72… Relative value of total peak area related to false positive amplification products
81 ... Allele A sample group
82… Sample group of heterozygote
83 ... Allele T specimen group

Claims (7)

A nucleic acid amplification method for obtaining an amplification product having a desired base length range including a single nucleotide polymorphism 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 designed to hybridize to the single nucleotide polymorphic site on the first strand at the 3 ′ end; and the target A second primer designed to hybridize to a site which comprises a sequence complementary to the base sequence of the second strand of the nucleic acid and forms a base pair with the single nucleotide polymorphism site on the second strand; By performing nucleic acid amplification using, and controlling the denaturation temperature in the nucleic acid amplification to 60 to 85 ° C. from at least the 5th cycle , dissociating the amplification product in the desired base length range into single strands, The nucleic acid amplification method, wherein the nucleic acid amplification method is used as a template for the next nucleic acid synthesis reaction. The amplification product of the desired base length range is a nucleic acid synthesized by hybridizing the 3 ′ ends of the first primer and the second primer to a single nucleotide polymorphism site on the target nucleic acid. 2. The nucleic acid amplification method according to 1. The nucleic acid amplification method according to claim 1 or 2 , wherein both the first primer and the second primer base length are 18 to 50. The nucleic acid amplification method according to any one of claims 1 to 3 , wherein melting temperatures of the first primer and the second primer are substantially the same. The nucleic acid amplification method according to any one of claims 1 to 4 , 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 the sequence of a single nucleotide polymorphism 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 designed to hybridize to the single nucleotide polymorphic site on the first strand at the 3 ′ end; and the target A second primer designed to hybridize to a site which comprises a sequence complementary to the base sequence of the second strand of the nucleic acid and forms a base pair with the single nucleotide polymorphism site on the second strand; And the denaturation temperature in the nucleic acid amplification is controlled to at least 60 to 85 ° C. from the fifth cycle to dissociate the amplification product into single strands, and the template for the next nucleic acid synthesis reaction And a nucleic acid amplification step characterized by comprising: analyzing the sequence of a single nucleotide polymorphism site on the target nucleic acid by detecting the amplification product. The method according to claim 6 , wherein the detection of the amplification product is performed by a biochemiluminescence reaction using pyrophosphate generated in response to nucleic acid amplification.

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