GSTM1 typing test method based on quantitative PCR technique

A detection method and technology, applied in the field of genetic detection, can solve the problems affecting the accurate determination of gene trait analysis, difficult heterozygous genotype, difficult experiment reliability, etc., to simplify the detection and result analysis process, avoid pollution, and prevent PCR The effect of product contamination

Inactive Publication Date: 2017-08-18
上海龙鼎医药科技有限公司
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Problems solved by technology

Although it is simple, it is easy to cause laboratory pollution because the electrophoresis operation must open the tube to add samples and electrophoresis, and it is difficult to ensure the reliability of the exper

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  • GSTM1 typing test method based on quantitative PCR technique

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Effect test

Embodiment 1

[0038] (1) Preparation of normal human genomic DNA for testing: take normal human oral and throat swabs, extract and prepare with Chlex100, and dilute the concentration of normal human genomic DNA to 10 ng / μl;

[0039] (2) Design and synthesis of PCR-specific primers and probes: Design and synthesize gene-specific primers SEQ1, SEQ2 and GSTM1-specific TAQman gene probe marker Fam according to the human GSTM1 gene sequence; design an internal reference gene-specific PCR primer SEQ4 based on the human ALbumin gene , SEQ5 and probe SEQ6 mark Vic;

[0040] (3) GSTM1 quantitative PCR detection:

[0041] 1) Primer-probe mix configuration:

[0042] 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.

[0043] 3) Quantitative PCR instrument: ABI7500

[0044] 4) Reaction conditions: 95°C, 2min; 95°C, 15s---60°C, 32s, 42 cycles.

[0045] (4) Gene verification result read:

[0...

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Abstract

The invention discloses a GSTM1 typing test method based on a quantitative PCR technique. The typing test method adopts GSTM1 gene-specific primers, SEQ1 and SEQ2, to amplify a GSTM1 gene segment, and meanwhile a GSTM1 gene-specific TAQman probe, SEQ3-Fam, is designed in an amplified region which is defined by the GSTM1 gene-specific primers; ALbumin-specific PCR primers, SEQ4 and SEQ5, are adopted to amplify an Albumin gene segment, and meanwhile an ALbumin gene-specific TAQman probe, SEQ6-Vic, is designed in an amplified region which is defined by the ALbumin gene-specific primers. The typing test method can effectively prevent PCR product pollution, avoid contamination by poisonous DNA colouring agents, greatly simplify test and result analyzing processes, and increase test throughput.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a GSTM1 typing detection method based on quantitative PCR technology. Background technique [0002] GSTM1 is an important metabolic enzyme involved in the transformation and metabolism of endogenous and exogenous substances, endowing metabolites with new biological properties. GSTM1 is polymorphic in humans, and GSTM1 deletion is a common genotype. There are differences in the transformation function of the individual geomaterials of different GSTM1 genotypes. GSTM1 genotyping is used in the study of various biological traits. Traditionally, since the GSTM1 deletion polymorphism can be detected by ordinary PCR, conventional gene-specific PCR and agarose electrophoresis detection methods are often used to confirm the GSTM1 genotype. Although it is simple, it is easy to cause laboratory pollution because the electrophoresis operation must be opened to add samples and elec...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2561/101
Inventor 杨随娟杨燕燕刘虎
Owner 上海龙鼎医药科技有限公司
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