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Primer for detecting polymorphism of human ALDH2 gene by TaqMan-MGB probe method and application of primer

A technology of gene polymorphism and probe method, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of incomplete fluorescence quenching, and achieve good quenching effect and polymorphism typing Precise, easy-to-operate results

Inactive Publication Date: 2017-06-13
CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the problem of incomplete fluorescence quenching of TaqMan probes, in 2000, ABI Corporation of the United States introduced a new TaqMan probe – MGB probe (miNO.r groove binder oligodeoxynucleotide conjugate). Group – the quenching group absorbs the energy of the reporter group and does not emit light, which greatly reduces the interference of the background signal

Method used

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  • Primer for detecting polymorphism of human ALDH2 gene by TaqMan-MGB probe method and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: DNA Extraction

[0033] 200 μL of peripheral blood was taken, and DNA was extracted according to the instructions of the whole blood genomic DNA extraction kit (purchased from Hangzhou Xinjie Biotechnology Co., Ltd.), and the DNA was extracted for subsequent experiments or stored at -20°C.

Embodiment 2

[0035] Amplification primers and TaqMan-MGB probes for detecting the rs671 polymorphism of the ALDH2 gene were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.:

[0036] Primer pairs for amplifying the ALDH2 gene:

[0037]F (SEQ ID NO.: 1): 5'-CCCTTTGGTGGCTACAAGATG-3',

[0038] R (SEQ ID NO.: 2): 5'-GCAGGTCCCACACTCACAGTT-3';

[0039] TaqMan-MGB probe for fluorescent quantitative typing of ALDH2 gene:

[0040] FAM (SEQ ID NO.: 3): 5'-AGGCATACACTGAAGT-3',

[0041] VIC (SEQ ID NO.: 4): 5'-AGGCATACACTAAAGTG-3'.

Embodiment 3

[0042] Embodiment 3: detection method

[0043] Instrument: Roche 480 fluorescent quantitative PCR detector, Thermo Legend Micro 21 high-speed centrifuge, Taicang Hualida Laboratory Equipment Company WH-866 vortex oscillator.

[0044] (1) With the genomic DNA that embodiment (1) obtains as template, utilize specific amplification primer and sensitive and stable TaqMan-MGB probe to carry out the detection of ALDH2 gene, specifically comprise the following steps:

[0045] (a) Preparation of PCR reaction solution: from the 2x PCR reaction mixture and each component in Example 2, melt at room temperature and put it on an ice box for later use. Within 10 minutes before sample addition, prepare PCR reaction solution Xμl according to the number of samples to be tested:

[0046] X=10μl PCR reaction solution+0.4μl F primer+0.4μl R primer+0.6μl FAM probe+0.3μl VIC probe

[0047] (b) Add 50ng of template DNA, add water to make up to a total volume of 20μL, and set up a positive standa...

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Abstract

The invention discloses a reagent kit for detecting polymorphism of a human ALDH2 (acetaldehyde dehydrogenase 2) gene by a TaqMan-MGB (minor groove binder) probe method. The reagent kit comprises the following nucleic acid sequences: (1) a primer pair for amplifying an rs671 polymorphism site of the ALDH2 gene, and (2) a TaqMan-MGB probe for detecting the rs671 polymorphism site of the ALDH2 gene, wherein a nucleotide sequence of the primer pair is shown as SEQ ID NO. 1 and SEQ ID NO. 2; a nucleotide sequence of the TaqMan-MGB probe is shown as SEQ ID NO. 3 and SEQ ID NO. 4; the SEQ ID NO. 3 is used for labeling an FAM signal; and the SEQ ID NO. 4 is used for labeling a VIC signal. The invention further discloses an application of a primer and the probe in detecting the polymorphism of the human ALDH2 gene. The primer has the advantages of accurate detection result, quickness, high efficiency, convenience and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a primer for detecting human ALDH2 gene polymorphism by a TaqMan-MGB probe method and an application thereof. Background technique [0002] The excessive enrichment of ethanol and acetaldehyde produced in the process of alcohol metabolism and the blockage of nitroglycerin metabolism often bring health risks to people, such as alcoholic liver disease, neurological disease, cardiovascular disease, esophageal cancer, gastric cancer and other diseases. Human acetaldehyde dehydrogenase (ALDH) is a key enzyme in alcohol metabolism. ALDH has 19 isoenzymes, of which ALDH2 is the most important, has dehydrogenase activity, and plays a major role in the oxidation of acetaldehyde in the human body (that is, it is directly related to the ability to metabolize alcohol in the body). ALDH2 mainly acts as a catalyst for the second-phase metabolism in the ethanol metabolic pat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6851C12Q1/6883C12Q2600/106C12Q2600/156
Inventor 任绪义张锋周韵
Owner CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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