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Method for detecting polymorphism of C677T site of human MTHFR gene

A technology for site polymorphism and human detection, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Low cost, reduced risk of false positives, and the effect of shortening the detection cycle

Pending Publication Date: 2021-01-12
合肥中科支众医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the 222nd amino acid of the protein encoded by the MTHFR gene is changed from alanine (Ala) to valine (Val) after the c.677 site C is changed to T, resulting in the thermostability and enzyme activity of the MTHFR enzyme reduce

Method used

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  • Method for detecting polymorphism of C677T site of human MTHFR gene
  • Method for detecting polymorphism of C677T site of human MTHFR gene
  • Method for detecting polymorphism of C677T site of human MTHFR gene

Examples

Experimental program
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Effect test

Embodiment 1

[0046] The method for extracting DNA from people's oral swab samples and detecting the polymorphism of the C677T site of the MTHFR gene comprises the following steps:

[0047] Step 1: According to the position on the genome of the MTHFR gene C677T site (rs1801133) in the NCBI database, design corresponding primers and probes. The specific sequences are as follows:

[0048]MTHFR gene C677T forward amplification primer: 5'-GCCTCAAAGAAAAGCTGCGT-3';

[0049] MTHFR gene C677T reverse amplification primer: 5'-TCTGAAGCACTTGAAGGAGAAGGT-3';

[0050] MTHFR gene 677C probe: 5'-FAM-ATGAAATCGGCTCCC-MGB-NFQ-3';

[0051] MTHFR gene 677T probe: 5'-VIC-ATGAAATCGACTCCC-MGB-NFQ-3'.

[0052] Step 2: Reagent preparation

[0053] (1) Self-designed primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd.,

[0054] (2) Magnetic bead method saliva & swab genomic DNA extraction kit (purchased from Yingruicheng),

[0055] (3) KAPA PROBE FAST Universal qPCR Kit (purchased from KAP...

Embodiment 2

[0073] The method for extracting DNA from a human peripheral blood cell sample and detecting the polymorphism of the C677T site of the MTHFR gene comprises the following steps:

[0074] Step 1: According to the position on the genome of the MTHFR gene C677T site (rs1801133) in the NCBI database, design corresponding primers and probes. The specific sequences are as follows:

[0075] MTHFR gene C677T forward amplification primer: 5'-GCCTCAAAGAAAAGCTGCGT-3';

[0076] MTHFR gene C677T reverse amplification primer: 5'-TCTGAAGCACTTGAAGGAGAAGGT-3';

[0077] MTHFR gene 677C probe: 5'-Cy5-ATGAAATCGGCTCCC-MGB-NFQ-3';

[0078] MTHFR gene 677T probe: 5'-ROX-ATGAAATCGACTCCC-MGB-NFQ-3'.

[0079] Step 2: Reagent preparation

[0080] (1) Self-designed primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd.,

[0081] (2) Peripheral blood genomic DNA extraction kit (purchased from Tiangen),

[0082] (3) Genotyping PCR reagent (probe method) (purchased from Tiangen).

...

Embodiment 3

[0095] The method for detecting the polymorphism of the C677T site of the MTHFR gene by searching and randomly obtaining four DNA samples analyzed as genotype TT from the results of the first-generation sequencing, comprising the following steps:

[0096] Step 1: According to the position on the genome of the MTHFR gene C677T site (rs1801133) in the NCBI database, design corresponding primers and probes. The specific sequences are as follows:

[0097] MTHFR gene C677T forward amplification primer: 5'-GCCTCAAAGAAAAGCTGCGT-3';

[0098] MTHFR gene C677T reverse amplification primer: 5'-TCTGAAGCACTTGAAGGAGAAGGT-3';

[0099] MTHFR gene 677C probe: 5'-FAM-ATGAAATCGGCTCCC-MGB-NFQ-3';

[0100] MTHFR gene 677T probe: 5'-HEX-ATGAAATCGACTCCC-MGB-NFQ-3'.

[0101] Step 2: Reagent preparation

[0102] (1) Self-designed primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd.,

[0103] (2) Genotyping PCR reagent (probe method) (purchased from KAPA).

[0104] Step 3: Pre...

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Abstract

The invention relates to the technical field of gene polymorphism detection, and particularly relates to a method for detecting mutation of a C677T site (rs1801133) of an MTHFR gene. Wild type and mutant type primers and probes are designed for the site, and a wild type target fragment and a mutant type target fragment of the site are amplified with a qPCR technology. Compared with other methods,the method has the advantages of being short in detection period, high in specificity, accuracy and flux, low in cost, visual and convenient in interpretation result, high in generalizability and thelike.

Description

technical field [0001] The invention belongs to the technical field of gene polymorphism detection, and specifically refers to a method for detecting the polymorphism of human MTHFR gene C677T site (rs1801133). Background technique [0002] Folic acid, also known as vitamin B9. Folic acid plays an important role in DNA synthesis, DNA methylation, etc. It is related to the normal development of embryos, health maintenance and the risk of various diseases. It is an indispensable nutrient for cell proliferation, tissue growth and body development. There are two reasons for the lack of folic acid in the body: one is insufficient folic acid intake, and the other is the low ability of the body to utilize folic acid (folic acid metabolic pathway disorder) due to genetic (gene) defects. Once folic acid deficiency or folic acid metabolic enzyme deficiency occurs in the body, it will lead to abnormal cell cycle, abnormal DNA and protein methylation reactions, abnormal synthesis of DN...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/686C12N15/11
CPCC12Q1/6858C12Q1/686C12Q2561/113C12Q2563/107C12Q2561/101
Inventor 刘彦哲万鲲李丹丹张姗姗段梦梦聂金福
Owner 合肥中科支众医学检验有限公司