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Methylated quantitative detection method of GSH2 (glutathione 2) gene

A methylation quantitative and detection method technology, applied in the field of GSH2 gene methylation detection, can solve the problems of restricting the use of MSP, and there is no quantitative detection method of GSH2 gene methylation, etc., to achieve simple operation, high sensitivity, The effect of strong accuracy

Inactive Publication Date: 2010-09-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

The key to reliable MSP lies in the primers. The primer design requires two known regions containing multiple fully methylated or unmethylated CpG sites, but there are not many CpG islands with the above characteristics, which limits the potential of MSP. use
This method has the following disadvantages: (1) This method can only be used for qualitative research, that is, it can only determine whether there is methylation; if quantification is required, other methods need to be used for further detection; Due to the difference in the amount of amplification products between methylated primers and unmethylated primers, it is necessary to strictly control the conditions and cycle number of the PCR reaction.
[0008] Currently, there is no quantitative detection method related to GSH2 gene methylation

Method used

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  • Methylated quantitative detection method of GSH2 (glutathione 2) gene
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  • Methylated quantitative detection method of GSH2 (glutathione 2) gene

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Embodiment 1

[0024]Example 1: Quantitative detection of methylation degree of GSH2 gene (GeneID: 170825) in clinical samples of pancreatic cancer patients

[0025] Sources of main reagents and instruments: QIAGEN EpiTect Bisufite KitTM kit, pMD18-T Vector, EX-taq enzyme purchased from Treasure Bioengineering (Dalian) Co., Ltd., ABITaqman Gene Expression Master Mix purchased from Applied Biosystems, ABI 7500 Real Time PCR The instrument was purchased from Applied Biosystems, USA, and the primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0026] Steps:

[0027] 1. Preparation of standard products using gene amplification and bioengineering methods

[0028] 1. Genomic DNA Extraction

[0029] DNA was extracted by conventional phenol / chloroform method.

[0030] (1) Put 50mg of pancreatic tissue into a 5ml centrifuge tube, add 700ul of lysis buffer to make a homogenate.

[0031] (2) Add proteinase K to a final concentration of 100ug / ml, and keep at 55°C overni...

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Abstract

The invention belongs to the technical field of biomedical sciences. Abnormal methylation of genes participates in the development of tumors. The invention aims at overcoming the defects of a methylation-sensitive restrictive endonuclease method, a bisulfite sequencing method and a methylation-specific PCR (Polymerase Chain Reaction) method and provides a methylated quantitative detection method of a GSH2 (glutathione 2) gene, which has simple and convenient operation and high senibilitiy. The method comprises the following steps of: taking pancreatic cancer tissues, extracting DNA and preparing an ACTB (Actin, Beta) standard curve sample, and preparing a GSH2 (Glutathione 2) standard curve sample by utilizing a full methylation template; extracting DNA of tissues to be detected, and carrying out chemical modification; designing methylation primers and probes aiming at CPG islands at a human GSH2 gene promoter region, designing BSP (Bisulfite) primers and probes aiming at CPG islands at a human reference gene ACTB promoter region, carrying out real-time quantitive PCR on DNA after bisulfite treatment by adopting a Taqman-MGB real-time quantitive PCR method, and calculating a quantitive value of the GSH2 gene methylation degree. The method is rapid, accurate and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting nucleic acid, in particular to a method for detecting methylation of the GSH2 gene. Background technique [0002] The occurrence and development of malignant tumors is a process in which multiple genes interact and influence each other. Abnormal methylation of CpG islands is an important mechanism for the occurrence and development of this gradual process, which includes two types: hypermethylation and hypomethylation. According to literature reports, abnormal methylation of multiple pancreatic cancer-related genes has been detected in pancreatic cancer tissue or pancreatic juice, while the proportion of methylation in normal tissues is very low, so the detection of methylation of pancreatic cancer-related genes , to achieve the purpose of early diagnosis of pancreatic cancer. For example, abnormal methylation of gene P16 (Virmani AK, et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李兆申黄浩杰高军杜奕奇龚燕芳施新岗王丽华顾俊骏许爱平
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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