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SNP nucleic acid mass spectrometric detection method for XPC gene rs2228001 locus

A technology of rs2228001-f and rs2228001-e, which is applied in the field of SNP nucleic acid mass spectrometry detection at the rs2228001 site of the XPC gene, can solve problems such as the decline of DNA repair ability, and achieve the effects of improving operability, ensuring accuracy, and strong reliability

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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  • Abstract
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  • Application Information

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Problems solved by technology

Defects in XPC function may result in decreased DNA repair capacity, leading to a range of diseases including tumors

Method used

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  • SNP nucleic acid mass spectrometric detection method for XPC gene rs2228001 locus
  • SNP nucleic acid mass spectrometric detection method for XPC gene rs2228001 locus
  • SNP nucleic acid mass spectrometric detection method for XPC gene rs2228001 locus

Examples

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experiment example

[0051] A method for detecting the SNP nucleic acid mass spectrometry of the rs2228001 site of the XPC gene, comprising the steps of:

[0052] S1: Perform the first round of PCR with the upstream primer rs2228001-F and downstream primer rs2228001-R to amplify the template DNA. The base sequences of the primers are as follows:

[0053] Upstream primer rs2228001-F: 5`-ACGTTGGATGAACTGGTGGGTGCCCCTCTA-3`;

[0054] Downstream primer rs2228001-R: 5`-ACGTTGGATGGCCTCAAAACCGAGAAGATG-3`;

[0055] The reaction system is as follows:

[0056]

[0057] The reaction conditions are as follows:

[0058] (1) Pre-denaturation: 95°C for 2 minutes;

[0059] (2) Amplification: 95°C for 30s, 56°C for 30s, 72°C for 60s, a total of 45 cycles;

[0060] (3) Extension: 72°C for 5min, 4°C∞;

[0061] 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 follows:

[0062] ...

experiment example 1

[0089] With the method provided by the present invention as the experimental group, with the method provided by the control example as the control group 1, with the conventional Taqman method as the control group 2, respectively to the XPC gene rs2228001 site standard substance of 15ng (comprising 5ng GT heterozygous, 5ng GG homozygous and 5ng TT 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 method provided by the present invention is more accurate and better than the existing method for the SNP typing detection of the XPC gene rs2228001 site, and it also shows that the denaturation-annealing internal cycle set ...

experiment example 2

[0092] Randomly select 26 healthy adult volunteers (18 to 60 years old) to collect blood samples and extract the whole blood genome, and perform rs2228001 SNP detection 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 2-5 As shown, a total of 22 samples obtained analyzable results, among which 11 samples were TT homozygous, 9 samples were GT heterozygous, and 2 samples were GG homozygous. It shows that there are more TT homozygotes at this SNP site, and GG homozygotes and G-T 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 3-5 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 an XPC gene rs2228001 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; and finally, the rs2228001 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 XPC gene rs2228001 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 rs2228001 site of XPC 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 XPC gene is located in the 25th region of chromosome 3, belongs to the xeroderma pigmentosa genome, and is an important DNA damage repair gene. As an evolutionarily conserved DNA repair enzyme, XPC is involved in DNA damage recognition and the initiation of NER (nucleotide excision re...

Claims

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Application Information

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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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