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MTHFR and MTRR gene polymorphism detection primer group and kit

A technology for gene polymorphism and primer detection, which is used in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc. It can solve cross-contamination of PCR products, insufficient enzyme digestion, and low reliability. problem, to achieve good amplification effect, high accuracy, and reduce pollution.

Active Publication Date: 2016-01-20
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there are many methods for the detection of MTHFR and MTRR gene polymorphisms. The simplest method is PCR-RFLP. False-negative or false-positive results due to overcutting, low reliability
Although the DNA sequencing method is the gold standard, the steps are cumbersome, the process is complicated, and cross-contamination between samples is prone to occur, resulting in sequencing failure. In addition, the price of the sequencer is beyond the tolerance of general clinical testing laboratories.
The high-resolution melting curve method is fast, simple, economical and practical, but it is controlled by the temperature of the instrument, and the false positive is high.

Method used

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  • MTHFR and MTRR gene polymorphism detection primer group and kit
  • MTHFR and MTRR gene polymorphism detection primer group and kit
  • MTHFR and MTRR gene polymorphism detection primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Extraction of clinical sample DNA

[0064] 1. EDTA anticoagulant

[0065] In this example, genomic DNA was extracted from EDTA anticoagulated blood and quantified as a template for PCR detection. The blood genomic DNA extraction kit (TIANampDNABloodMiniKit) from TIANGEN Company was used, and the operation was performed according to the instructions of the kit, as detailed below.

[0066] 1. Processing blood material:

[0067] a. When the volume of the blood sample is less than 200 μL, add buffer GS to make up the volume to 200 μL, and then proceed to the next step of the experiment (if the volume of the blood sample is 200 μL, the next step of the experiment can be carried out directly without adding GS).

[0068] b. When the volume of the blood sample exceeds 200 μL, it needs to be treated with cell lysate CL. The specific steps are as follows: add 1 to 2.5 times the volume of cell lysate CL to the sample, mix it upside down, and centrifuge at 10,000 rp...

Embodiment 2

[0095] Example 2: Real-time fluorescent PCR method to amplify clinical sample DNA

[0096] This example uses the primers and probes provided by the present invention to amplify the DNA sample extracted in Example 1. Two parts of genomic DNA were diluted with TE diluent to a concentration of 1 ng / μL, and amplified on an ABI7300 fluorescent quantitative PCR instrument with the kit of the present invention.

[0097] Fluorescent quantitative PCR reaction system is:

[0098] PCR reaction solution:

[0099] MgCl 2 : 2.0~5.0mmol

[0100] dNTPs: 0.2~0.8mmol

[0101] Each primer: 0.1~1.0μmol

[0102] Each probe: 0.1~1.0μmol

[0103] ABIFastmasterpremix: 6~10μL

[0104] Template: 10 μL

[0105] Total volume: 40 μL.

[0106] The preferred PCR reaction tubes are:

[0107] The first stage: 95°C for 5 minutes;

[0108] The second stage: 95°C for 5s, 58°C for 30s, 10 cycles;

[0109] The third stage: 95°C for 5s, 58°C for 30s, 72°C for 30s, 35 cycles;

[0110] Phase III: At 35 c...

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Abstract

The invention discloses an MTHFR and MTRR gene polymorphism detection primer group and a kit. Mutation primers and probes aiming at three genetic loci have the advantages of being high in specificity and sensitivity. The prepared kit can detect MTHFR677, MTHFR1298 and MTRR66 gene polymorphism conditions, operation is simple, the experimental period is short, and the primer group and the kit are safe, free of toxicity, low in cost and suitable for clinical large-scale use and popularization.

Description

technical field [0001] The invention relates to the technical field of gene polymorphism detection, in particular to a primer set and a kit for detecting MTHFR and MTRR gene polymorphisms. Background technique [0002] SNP, Single Nucleotide Polymorphisms (full name Single Nucleotide Polymorphisms), refers to the genetic markers formed by the variation of a single nucleotide on the genome (including substitutions, transversions, deletions and insertions), with a large number and rich polymorphisms. From a theoretical point of view, each SNP site can have 4 different variants, but in fact there are only two of them, namely conversion and transversion, and the ratio of the two is 1:2. SNPs appear most frequently on CG sequences, and most of them are converted from C to T. The reason is that C in CG is often methylated and becomes thymine after spontaneous deamination. Generally speaking, a SNP refers to a single nucleotide variation with a variation frequency greater than 1%....

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 胡利红段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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