Method for carrying out typing detection of A66G polymorphic site of MTRR gene

A technology of polymorphic sites and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of complex operation, cross-contamination of samples, low sensitivity, etc., and achieve good specificity and repeatability High, high-sensitivity effects

Inactive Publication Date: 2017-08-11
上海龙鼎医药科技有限公司
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Problems solved by technology

[0004] At present, the most commonly used genetic polymorphism detection method is the sequencing method. The advantage of this method is that it can perform high-throughput multi-site detection at the same time, but its operation is complicated, time-consuming and low-sensitivity. Cross-contamination between samples is prone to occur and rapid detection of samples cannot be realized; the high-resolution melting curve method is fast, simple, economical and practical, but it has high requirements for instruments, and it needs to be used with high-resolution software and temperature-sensitive machines. Difficulties in clinical promotion

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  • Method for carrying out typing detection of A66G polymorphic site of MTRR gene
  • Method for carrying out typing detection of A66G polymorphic site of MTRR gene
  • Method for carrying out typing detection of A66G polymorphic site of MTRR gene

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Embodiment

[0033] (1) Preparation of normal human genomic DNA for testing:

[0034] Oropharyngeal swabs of normal people were taken, prepared by Chlex100 extraction, and the concentration of normal human genome DNA was diluted to 10ng / μl.

[0035] (2) Design and synthesis of PCR-specific primers and probes:

[0036] Synthetic gene-specific primers SEQ1, SEQ2 and MTRR-specific TAQman gene probe markers SEQ3-FAM-A and SEQ4-VIC-G were designed according to the human MTRR gene sequence.

[0037] (3) MTRR quantitative PCR detection:

[0038] 1) Primer-probe mix configuration:

[0039] 2) Reaction system: 1.5 μl of primer-probe mixture, 12.5 μl of 2-fold TIANtoughGenotypingqPCCRPreMix (Probe), 1.5 μl of standard template, and 9.5 μl of ddH2O.

[0040] 3) Quantitative PCR instrument: ABI7500.

[0041] 4) Reaction conditions: 95°C, 2min; 95°C, 15s——60°C, 32s, 42 cycles.

[0042] (4) Gene verification result read:

[0043] see figure 1 , figure 1 The middle abscissa is the cycle number ma...

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Abstract

The invention discloses a method for carrying out typing detection of an A66G polymorphic site of an MTRR gene. An MTRR gene segment is amplified by adopting MTRR gene-specific primers SEQ1 and SEQ2; and meanwhile, MTRR gene-specific TAQman probes SEQ3-FAM-A and SEQ 4-VIC-G are designed in an amplification area defined by an MTRR gene-specific primer. The method is used for judging gene SNP site typing on the basis of gene-specific PCR combined with the TAQman probes; and by adopting MTRR-specific gene amplification and the TAQman probes, target gene typing detection is carried out on the basis of a quantitative PCR method of the TAQman probes, so that the method has the characteristics of being simple, fast, high in repeatability, high in sensitivity and good in specificity.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a typing detection method for the A66G polymorphism site of the MTRR gene. Background technique [0002] Folic acid belongs to the water-soluble B vitamins, plays an important role in the biosynthesis of nucleic acids and proteins, and is the material basis for cell proliferation and body development. China is one of the countries with a high incidence of birth defects in newborns, and folic acid deficiency is the main cause of birth defects. There are two main reasons for the lack of folic acid in the body: one is insufficient intake of folic acid, and the other is the low efficiency of the body's use of folic acid due to genetic defects, that is, folic acid metabolism disorder. [0003] The full name of MTRR is methionine synthase reductase, which is the key enzyme to maintain the activity of methionine synthase. Methionine synthase plays an important role in folic acid...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2561/101
Inventor 杨燕燕刘虎杨随娟
Owner 上海龙鼎医药科技有限公司
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