CN114214414A - SNaPshot multiplex analysis kit for detecting SNP (Single nucleotide polymorphism) of ALL (ALL related gene) and application of snaPshot multiplex analysis kit - Google Patents
SNaPshot multiplex analysis kit for detecting SNP (Single nucleotide polymorphism) of ALL (ALL related gene) and application of snaPshot multiplex analysis kit Download PDFInfo
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Abstract
The invention relates to a SNaPshot multiplex analysis kit aiming at multiple ALL related SNP sites and application thereof. The detection result of the SNaPshot multiplex analysis kit is consistent with that of a sanger sequencing method, and the common SNP loci of 5 genes of ALL can be accurately detected. Compared with a sanger sequencing method, the result analysis and the experimental process of the SNaPshot multiplex analysis kit are faster and more efficient. Therefore, the detection efficiency of the ALL gene polymorphism is improved, and the detection cost is reduced.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a SNaPshot multiplex analysis kit for detecting SNP (Single nucleotide polymorphism) of ALL (ALL related genes) and application thereof.
Background
With the discovery of SNP sites of a plurality of genes, the relationship between drug individuation and gene polymorphism of children suffering from Acute Lymphoblastic Leukemia (ALL) is receiving increasing attention, and the gene polymorphism influencing the metabolism of chemotherapy drugs of children suffering from ALL has been used as a prediction factor of adverse reactions and prognosis of ALL chemotherapy.
Sequencing technology is the most important detection means in molecular biology, is a gold standard for detecting gene polymorphism and gene mutation, and is also a reference standard for other numerous technical methods. The sequencing technology has the advantages that the gene sequence of a sample is directly determined to obtain the accurate condition of gene variation, and the method can carry out multi-site mutation detection or unknown mutation detection on a certain section of DNA sequence, and is widely applied to various fields of tumor targeted drug individualized treatment detection, genetic disease gene mutation detection, blood disease gene mutation detection, HLA typing and matching, pathogenic microorganism drug resistance detection and the like. The first generation of DNA sequencing technology used the chain termination method pioneered by Sanger and Coelson in 1975 or the chemical method (chain degradation) invented by Maxam and Gilbert. The principle of the commonly used Sanger sequencing (dideoxy end-termination) is that a fluorescently labeled ddNTP is incorporated into a dNTP, and due to the random incorporation of the ddNTP, the PCR product starts at the first base after the primer, and there is a possibility that a ddNTP is present at every position. Since ddNTP lacks the 3' -OH required for chain extension, the extension of the chain is selectively terminated at G, A, T or C. Unlike ordinary PCR, such PCR products do not form an electrophoretic band, but a set of hundreds or thousands of fragments differing in length by one base. They have a common starting point, terminate at different nucleotides, and each base has the same probability of being terminated. And carrying out capillary electrophoresis on the obtained fragments with different sizes, and finally obtaining the sequence of the target fragment by collecting and splicing the fluorescence signals. It is characterized by high reading length; detecting in flux; the detection sensitivity is low; the single-hole is low in cost, is suitable for sequencing a small number of loci of a few genes, and is widely applied to clinical detection and scientific research.
The SNaPshot multiplex analysis system is also called as a small sequencing technology, and is a method based on primer single base extension, in a reaction system containing sequencing enzyme, four kinds of fluorescence labeled ddNTP, extension primers with different lengths close to the 5' end of a polymorphic site and a PCR product template, the primer extension is terminated after one base, after glue running by an ABI sequencer, the type of the incorporated base can be known according to the color of a peak, and thus the genotype of a sample is determined. And designing extension primers with different lengths aiming at different SNP sites to realize the typing of a plurality of SNPs in one reaction system. The SNaPshot has the characteristic that the single base extension of ddNTP in Sanger sequencing is terminated, and has the property of high fragment analysis flux.
Disclosure of Invention
Based on the background technology, the technical problem to be solved by the invention is to provide a SNaPshot multiplex assay kit for detecting ALL related gene SNP and application thereof, so that the detection efficiency of ALL gene polymorphism is improved, and the detection cost is reduced.
First, the SNaPshot multiplex assay kit provided by the invention comprises a multiplex amplification primer set and a multiplex sequencing primer set.
Preferably, the kit comprises 2 Vazyme mix, SNaPshot mix and ddH2O。
Secondly, the invention provides a SNaPshot multiplex amplification primer group for detecting the SNP of the ALL related gene, wherein the SNP of the ALL related gene and the primer sequences are shown in the following table:
secondly, the invention also provides a working solution of the SNaPshot multiplex amplification primer group for detecting the SNP of the ALL related gene, and the specific configuration of the working solution is shown as the following table: wherein the concentrations of the upstream primer and the downstream primer are both 100 MuM,
on the other hand, the invention also provides a SNaPshot multiplex amplification PCR reaction system for detecting the SNP of the ALL related gene, which is specifically shown in the following table:
| ddH2O | 6μL |
| working solution of multiplex PCR amplification primers | 2μL |
| Sample DNA (20-30ng/ul) | 2μL |
| 2*Vazyme mix | 10uL |
| TotaL | 20μL |
In another aspect, the present invention further provides a SNaPshot multiplex amplification PCR reaction program for detecting ALL-related gene SNP, which is specifically shown in the following table:
in another aspect, the invention also provides a SNaPshot multiplex sequencing primer for detecting the SNP of the ALL related gene, wherein the specific sequence of the sequencing primer is shown in the following table:
on the other hand, the invention also provides a working solution of the SNaPshot multiplex sequencing primer group for detecting the SNP of the ALL related gene, and the specific configuration of the working solution is shown in the following table: wherein the concentration of each sequencing primer is 100 mu M,
on the other hand, the invention also provides a SNaPshot multiplex sequencing PCR reaction system for detecting the SNP of the ALL related gene, and the specific configuration of the PCR reaction system is shown in the following table:
| ddH2O | 1μL |
| working solution of multiplex PCR sequencing primer | 1μL |
| Multiplex PCR purification | 3μL |
| SNaPshot mix | 5uL |
| TotaL | 10μL |
In another aspect, the present invention further provides a SNaPshot multiplex sequencing PCR reaction program for detecting ALL-related gene SNP, wherein the specific configuration of the PCR reaction program is shown in the following table:
in another aspect, the present invention also provides a SNaPshot multiplex assay method for detecting the above ALL-associated gene SNP, comprising the steps of:
1) releasing nucleic acid of a sample to be detected;
2) performing multiple PCR amplification on the nucleic acid obtained in the step 1) by using the multiple amplification primer group; the amplification system and amplification procedure are as described above;
3) purifying the amplification product, and then amplifying the purified amplification product obtained in the step 2) by using the multiple sequencing primer group to obtain a product with a fluorescent label; the PCR reaction system and reaction procedure are as described above;
4) and (4) detecting the purified fluorescence labeling product on a machine, and analyzing to obtain a result.
Preferably, in the present invention, the sample for detection may be whole blood, peripheral blood, buccal swab, pharyngeal swab, etc., but is not limited thereto;
preferably, in the present invention, the nucleic acid concentration of the sample to be tested is at least 20ng/ul, and the purity A260/A280 is at least 1.5.
In another aspect, the invention also provides an application of the multiple amplification primer set or the multiple sequencing primer set in preparation of a SNaPshot multiplex analysis kit or a reagent for detecting the SNP of the ALL-related gene.
The invention has the beneficial effects that: the invention develops the SNaPshot multiplex analysis kit aiming at a plurality of ALL related SNP sites for the first time, thereby improving the detection efficiency of ALL gene polymorphism and reducing the detection cost.
Drawings
FIG. 1 is a single well primer test gel (from left to right, sample 1, sample 2, sample 3 in that order);
FIG. 2 is a glue pattern after amplification of the multiplex PCR primers (marker, sample 1, sample 2, sample 3 in order from left to right);
FIG. 3 is a diagram showing the analysis of the results after multiplex sequencing (sample 1, sample 2, and sample 3 in this order from top to bottom);
FIG. 4 is a graph showing the result of sanger sequencing of sample 1 (SLC 01B1-TT, TPMT-10-AA, MTHFR-1298-AC, NUDT15-3-CC, CEP72-CC, TPMT-7-GG, MTHFR-677-CC, TPMT-5-GG in this order from top to bottom);
FIG. 5 is a graph showing the result of sanger sequencing of sample 2 (SLC 01B1-TT, TPMT-10-AA, MTHFR-1298-AA, NUDT15-3-CC, CEP72-CC, TPMT-7-GG, MTHFR-677-TC, TPMT-5-GG from top to bottom);
FIG. 6 is a graph showing the result of sanger sequencing of sample 3 (SLC 01B1-CC, TPMT-10-AG, MTHFR-1298-AA, NUDT15-3-CC, CEP72-CC, TPMT-7-GG, MTHFR-677-TC, TPMT-5-GG in this order from top to bottom).
Detailed Description
The principles and features of this invention are described below in conjunction with specific embodiments, the examples given are intended to illustrate the invention and are not intended to limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1A SNaPshot multiplex assay kit for detecting multiple ALL-associated SNP sites
A SNaPshot multiplex assay kit for detecting multiple ALL-associated SNP sites, comprising a multiplex amplification primer set and a multiplex sequencing primer set.
Wherein the sequences of the multiplex amplification primer set are shown in the following table:
TABLE 1.5 Gene 8 SNP sites multiplex amplification primer sequences
The specific configuration of the working solution of the multiplex amplification primer set is shown in the following table: wherein the concentrations of the upstream and downstream primers are 100. mu.M.
TABLE 2.5 Gene multiplex amplification primer formulation for 8 SNP sites
Wherein the dilution mode in the table is 166.8 μ L ddH2O + (2.4+2+2+1.4+0.8+4+2+2) μ L of each F represents, 166.8 μ L of ddH2O+2.4μLMTHFR-677F+2μLMTHFR-1298F+2μLCEP72F+1.4μLNUDT15F+0.8μLSLCO1B1F+4μLTPMT-10F+2μLTPMT-7F+2μLTPMT-5F。
The multiplex PCR reaction system of the multiplex amplification primer set is shown in the following table:
TABLE 3.5 multiplex PCR amplification System for 8 SNP sites of genes
| ddH2O | 6μL |
| Working solution of multiplex PCR amplification primers | 2μL |
| Sample DNA (20-30ng/ul) | 2μL |
| 2*Vazyme mix | 10uL |
| TotaL | 20μL |
The procedure for multiplex PCR reaction of the above multiplex amplification primer set is shown in the following table:
TABLE 4.5 multiplex PCR amplification procedure for 8 SNP sites of genes
Wherein, the specific sequence of the multiple sequencing primer is shown in the following table:
TABLE 5.5 Gene 8 SNP sites multiple sequencing primer sequences
The specific configuration of the working solution of the multiplex sequencing primer set is shown in the following table: wherein each sequencing primer concentration is 100. mu.M.
TABLE 6.5 Gene multiplex sequencing primer formulation for 8 SNP sites
The specific configuration of the PCR reaction system of the above multiplex sequencing primer set is shown in the following table:
TABLE 7.5 multiple sequencing reaction System for 8 SNP sites of genes
| ddH2O | 1μL |
| Working solution of multiplex PCR sequencing primer | 1μL |
| Multiplex PCR purification | 3μL |
| SNaPshot mix | 5uL |
| TotaL | 10μL |
The specific configuration of the PCR reaction program of the above multiplex sequencing primer set is shown in the following table:
TABLE 8.5 Gene 8 SNP sites multiplex sequencing reaction program
Example 2A SNaPshot multiplex assay for detecting the SNP of the ALL-related Gene described above
A SNaPshot multiplex assay method for detecting SNPs in the ALL-associated gene as described above, comprising the steps of:
1) releasing nucleic acid of a sample to be detected; the nucleic acid concentration of the sample to be detected is at least 20ng/ul, and the purity A260/A280 is at least 1.5.
2) Performing multiplex PCR amplification on the nucleic acid obtained in step 1) using a multiplex amplification primer set (Table 1); the amplification system and amplification procedure are described in tables 2 and 3, respectively;
3) purifying the amplification product, and then amplifying the purified amplification product obtained in the step 2) by using a multiple sequencing primer group (table 5) to obtain a product with a fluorescent label; the PCR reaction systems and reaction procedures are described in tables 7 and 8;
4) and (4) detecting the purified fluorescence labeling product on a machine, and analyzing to obtain a result.
Wherein the detection samples are respectively selected from samples 1-3 containing the following known SNP locus information, and the specific SNP locus information is shown in the following table:
TABLE 9 SNP site information of samples to be tested
Comparative example 1 detection of samples 1-3 by Sanger sequencing
The feasibility of the experiment of the invention was verified by detecting the SNP site information of the ALL-related gene in samples 1-3 using the sanger sequencing method known in the art.
The experimental results are as follows: as shown in FIGS. 1 to 6, compared with comparative example 1, the detection result of the SNaPshot multiplex assay kit of the present invention is consistent with that of the sanger sequencing method, and accurate detection of common SNP sites of 5 genes of ALL can be achieved. Compared with a sanger sequencing method, the result analysis and the experimental process of the SNaPshot multiplex analysis kit are faster and more efficient.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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ttttttttcc ctctatttag tcatttgaaa acataattta agtgtaaatg tatgatttta 60
tgcaggttt 69
Claims (10)
1. A SNaPshot multiplex amplification primer group for detecting SNP of an ALL related gene is characterized in that the specific sequence of the SNaPshot multiplex amplification primer group of the SNP of the ALL related gene is shown as SEQ ID NO. 1-16.
2. The working solution of the multiplex amplification primer set according to claim 1, wherein the specific configuration of the working solution is as shown in the following table: wherein the concentrations of the upstream primer and the downstream primer are both 100 mu M
3. A SNaPshot multiplex amplification PCR reaction system adapted for use according to any of claims 1 or 2, wherein the PCR reaction system is specified in the following table:
。
4. A SNaPshot multiplex amplification PCR reaction program adapted for use according to any of claims 1 or 2, wherein the PCR reaction program is as specified in the following table:
5. a SNaPshot multiplex sequencing primer for detecting SNP of ALL related genes is characterized in that the specific sequence of the sequencing primer is shown as SEQ ID NO. 17-24.
6. The working solution of the multiplex sequencing primer set according to claim 5, wherein the specific configuration of the working solution is as shown in the following table: wherein each sequencing primer concentration is 100 mu M
7. A SNaPshot multiplex sequencing PCR reaction system adapted to any one of claims 5 or 6, wherein the specific configuration of the PCR reaction system is as shown in the following Table:
。
8. A PCR reaction procedure adapted for the SNaPshot multiplex sequencing according to any one of claims 5 or 6, characterized in that it is as shown in the following table:
9. a SNaPshot multiplex assay kit for detecting a plurality of ALL-associated SNP sites, comprising the multiplex amplification primer set of any one of claims 1-4 and/or the multiplex sequencing primer set of any one of claims 5-8.
10. Use of the multiplex amplification primer set according to any one of claims 1 to 4 and/or the multiplex sequencing primer set according to any one of claims 5 to 8 for the preparation of a SNaPshot multiplex assay kit or reagents for the detection of ALL-related gene SNPs, characterized in that it comprises the following steps:
1) releasing nucleic acid of a sample to be detected;
2) performing multiplex PCR amplification on the nucleic acid obtained in step 1) using the multiplex amplification primer set according to any one of claims 1 to 4; wherein the amplification system and the amplification procedure are as described in claims 3 and 4;
3) purifying the amplification product, and then amplifying the purified amplification product obtained in the step 2) by using the multiple sequencing primer set of any one of claims 5 to 8 to obtain a product with a fluorescent label; the PCR reaction system and the reaction procedure are as described in claims 7 and 8;
4) and (4) detecting the purified fluorescence labeling product on a machine, and analyzing to obtain a result.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671739A (en) * | 2009-10-19 | 2010-03-17 | 广州益善生物技术有限公司 | Specific sequence, liquid phase chip and method for SNP detection of TPMT gene |
| CN103224980A (en) * | 2013-01-24 | 2013-07-31 | 武汉康圣达医学检验所有限公司 | Primer groups for detecting chemotherapeutic drug-related single nucleotide polymorphism (SNP) sites of methylene tetrahydrofolate reductase (MTHFR) gene and detection method therefor |
| CN106350586A (en) * | 2016-08-30 | 2017-01-25 | 武汉康圣达医学检验所有限公司 | Method for determining gene mutation site of hemophagocytic syndrome |
| CN110257523A (en) * | 2019-07-22 | 2019-09-20 | 上海市胸科医院 | A kind of primer sets and detection method detecting chemotherapeutical medicine curative effect and side effect related SNP |
| CN110512003A (en) * | 2019-09-24 | 2019-11-29 | 上海科新生物技术股份有限公司 | For detecting the detection kit and its application method of NUDT15 gene pleiomorphism |
| CN111235264A (en) * | 2020-02-20 | 2020-06-05 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human TPMT gene and NUDT15 gene |
-
2021
- 2021-12-27 CN CN202111619654.9A patent/CN114214414A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671739A (en) * | 2009-10-19 | 2010-03-17 | 广州益善生物技术有限公司 | Specific sequence, liquid phase chip and method for SNP detection of TPMT gene |
| CN103224980A (en) * | 2013-01-24 | 2013-07-31 | 武汉康圣达医学检验所有限公司 | Primer groups for detecting chemotherapeutic drug-related single nucleotide polymorphism (SNP) sites of methylene tetrahydrofolate reductase (MTHFR) gene and detection method therefor |
| CN106350586A (en) * | 2016-08-30 | 2017-01-25 | 武汉康圣达医学检验所有限公司 | Method for determining gene mutation site of hemophagocytic syndrome |
| CN110257523A (en) * | 2019-07-22 | 2019-09-20 | 上海市胸科医院 | A kind of primer sets and detection method detecting chemotherapeutical medicine curative effect and side effect related SNP |
| CN110512003A (en) * | 2019-09-24 | 2019-11-29 | 上海科新生物技术股份有限公司 | For detecting the detection kit and its application method of NUDT15 gene pleiomorphism |
| CN111235264A (en) * | 2020-02-20 | 2020-06-05 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting polymorphism of human TPMT gene and NUDT15 gene |
Non-Patent Citations (5)
| Title |
|---|
| RIHWA CHOI ET AL.: "Pathway genes and metabolites in thiopurine therapy in Korean children with acute lymphoblastic leukaemia", BR J CLIN PHARMACOL., vol. 85, no. 7, pages 1585 - 1597, XP071603418, DOI: 10.1111/bcp.13943 * |
| 叶启东 等: "儿童急性淋巴细胞白血病的个体化治疗――TPMT和MTHFR基因多态性", 中国实用儿科杂志, no. 4, pages 51 - 56 * |
| 吕慧 等: "亚甲基四氢叶酸还原酶基因多态性与儿童急性淋巴细胞白血病风险的相关性研究", 临床儿科杂志, vol. 29, no. 5, pages 2 - 1 * |
| 郭璐莹 等: "MDR1基因外显子26多态性与急性淋巴细胞白血病的系统评价", 中国煤炭工业医学杂志, no. 5, pages 76 - 80 * |
| 高萍等: "SLCO1B1 基因多态性对甲氨蝶呤治疗的影响", 中国临床药理学杂志, vol. 30, no. 8, pages 2 * |
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