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SNP nucleic acid mass spectrometric detection method for PLCE1 gene rs2274223 locus

A rs2274223-f, rs2274223-r technology, applied in the field of SNP nucleic acid mass spectrometry detection at the rs2274223 site of the PLCE1 gene, achieves the effects of strong reliability, guaranteed accuracy, and improved operability

Inactive Publication Date: 2018-06-01
沃森克里克(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the risk of gastric cancer, esophageal cancer and other diseases of the AG type is 1.5 times that of the AA type, while the GG type will increase this risk to 1.9 times

Method used

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  • SNP nucleic acid mass spectrometric detection method for PLCE1 gene rs2274223 locus
  • SNP nucleic acid mass spectrometric detection method for PLCE1 gene rs2274223 locus
  • SNP nucleic acid mass spectrometric detection method for PLCE1 gene rs2274223 locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0051] A method for detecting SNP nucleic acid mass spectrometry at the rs2274223 site of the PLCE1 gene, comprising the following steps:

[0052]S1: Perform the first round of PCR with the upstream primer rs2274223-F and downstream primer rs2274223-R to amplify the template DNA. The base sequences of the primers are as follows:

[0053] Upstream primer rs2274223-F: 5`-ACGTTGGATGCAAAGCCCATCCATCGAAAC-3`;

[0054] Downstream primer rs2274223-R:5`-ACGTTGGATGGATTCTCTGAGCAGTTACCG-3`;

[0055] The reaction system is as follows:

[0056]

[0057]

[0058] The reaction conditions are as follows:

[0059] (1) Pre-denaturation: 95°C for 2 minutes;

[0060] (2) Amplification: 95°C for 30s, 56°C for 30s, 72°C for 60s, a total of 45 cycles;

[0061] (3) Extension: 5min at 72°C, ∞ at 4°C;

[0062] S2: Dephosphorylate the amplified product obtained in step S1 to obtain a dephosphorylated fragment, and the SAP mixed liquid system used in the dephosphorylation operation is as follow...

experiment example 1

[0089] The method provided by the present invention is used as the experimental group, the method disclosed in Patent Publication No. 105238853A is used as the control group 1, and the conventional Taqman method is used as the control group 2, and the rs2274223 locus standard of the PLCE1 gene of 15 ng (including 5 ng GA hybrid combined, 5ng GG homozygous and 5ng AA homozygous) were detected, and the typing and quantitative results of each group of experiments were compared.

[0090] Such as figure 1 As shown, the typing results of the experimental group and the control group 1 were better than those of the control group 2, and the quantitative results of the experimental group were higher than those of the control group 1 and closer to the standard content. It shows that the SNP detection of the rs2274223 site of the PLCE1 gene by the method provided by the present invention is more accurate and more effective than the existing method, and it also shows that the denaturation-...

experiment example 2

[0092] Randomly select 26 healthy adult volunteers (18-60 years old) to collect blood samples and extract the whole blood genome, and perform SNP detection at rs2274223 on 26 samples according to the method provided in the examples, and analyze the Mass spectrometry results were analyzed.

[0093] Experimental results such as Figure 2-4 As shown, a total of 22 samples obtained analyzable results, of which 9 samples were heterozygous for GA and 13 samples were homozygous for AA. It shows that there are more AA homozygotes at this SNP site, and G-A heterozygotes are also detected. figure 2 The sample points of different genotypes are separated from each other and have no intersection with each other. Figure 3-4 The peak types and separation conditions of different types in the test are good, indicating that the primers provided by the present invention have good specificity, high sensitivity, and accurate typing results.

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Abstract

The invention provides an SNP nucleic acid mass spectrometric detection method for a PLCE1 gene rs2274223 locus. The method comprises the steps that: first, a fragment containing the SNP locus is amplified through the first-step PCR reaction, instead of separately amplifying the SNP locus to improve operability of an sample and accuracy of follow-up operation; dephosphorylation of an amplified product obtained in step S1 by SAP prevents connection of the 5' end and the 3' end of a DNA molecule, and the DNA molecule remains linear condition until subsequent steps are ready to ensure follow-up accuracy of an experiment; finally, the rs2274223 locus is conducted single-base extension through the step S3, denaturation-annealing internal loop is used for making DNA double-strands fully uncoiling and separated, so as to achieve accurate classification of different SNP. Through the method, SNP typing information of the PLCE1 gene rs2274223 locus can be quickly and accurately obtained. The operation is simple and reliability is strong, and the method can provide reliable reference information for subsequent related researches.

Description

technical field [0001] The invention belongs to the technical field of SNP detection, in particular to a mass spectrometry detection method for SNP nucleic acid at the rs2274223 site of PLCE1 gene. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans. Accounts for more than 90% of all known polymorphisms. SNPs exist widely in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. [0003] The PLCE1 gene is a gene for expressing phospholipase Cε-1. It is located on chromosome 10 and is found in many organs such as heart, kidney, adrenal gland, brain, liver, lung, spleen, skeletal muscle, prostate, thyroid, neutrophils and platelets. There are different levels of expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2533/101C12Q2531/113C12Q2565/627
Inventor 聂宵林茂俊张鹏刘晓霞白杨杨
Owner 沃森克里克(北京)生物科技有限公司
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