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Quantitative detection method of HPP1 gene methylation

A technology of methylation quantification and detection method, which is applied in the field of HPP1 gene methylation detection, can solve the problems of complexity, large amount of samples, and restrictions on the use of MSP, and achieve high sensitivity, high accuracy, and easy operation Effect

Inactive Publication Date: 2010-03-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

This method has the following disadvantages: (1) Since the CG is not limited to the CCGG sequence, the CG not in the sequence will be ignored; (2) only when the methylation status of the key sites related to transcription is detected, the The results of the detection method are meaningful; (3) Relatively speaking, the Southern method is more complicated and requires a large amount of sample; (4) There is a problem of false positives caused by incomplete digestion of the enzyme; (5) It is not suitable for mixing sample
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.

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  • Quantitative detection method of HPP1 gene methylation
  • Quantitative detection method of HPP1 gene methylation
  • Quantitative detection method of HPP1 gene methylation

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Embodiment 1

[0021] Example 1: Quantitative detection of methylation degree of HPP1 gene (GeneID: 23671) in clinical samples of patients

[0022] Sources of main reagents and instruments: QIAGEN EpiTect Bisufite KitTM kit, pMD18-TVector and EX-taq enzymes were purchased from Treasure Bioengineering (Dalian) Co., Ltd., ABI Taqman GeneExpression Master Mix was purchased from Applied Biosystems, Inc., and ABI 7500 Real Time PCR instrument Primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd. in Applied Biosystems, USA.

[0023] 1. Preparation of standard products using gene amplification and bioengineering methods

[0024] 1. Genomic DNA Extraction

[0025] DNA was extracted by conventional phenol / chloroform method.

[0026] (1) Put 50mg of tissue into a 5ml centrifuge tube, add 700ul of lysis buffer to make a homogenate.

[0027] (2) Add proteinase K to a final concentration of 100ug / ml, and keep at 55°C overnight until the solution becomes clear.

[0028] (3) ...

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Abstract

The invention belongs to the technical field of biomedicine. The abnormal methylation of the gene participates in the occurrence and the development of tumors, therefore, the invention aims at overcoming the defects of a methylation sensitive restriction enzyme method, a bisulfite sequencing method and a methylation specificity PCR method, and providing a quantitative detection method of HPP1 genemethylation with convenient operation and high sensitivity. The method comprises the steps of: taking pancreatic cancer tissue; extracting NDA to prepare a standard curve sample of ACTB; using a fullmethylated template to prepare a standard curve sample of HPP1; extracting the DNA of tissue to be tested, and chemically modifying; designing methylated primer and probe by aiming at the human HPP1gene promoter area CPG island; designing BSP primer and probe by aiming at the human reference gene ACTB promoter area CPG island; conducting real-time and quantitative polymerase chain reaction (PCR)to the DNA of processed sulfite by means of Taqman-MGB real-time quantitative PCR method; and counting the quantitative value of the HPP1 gene methylation degree. The method is fast and accurate, andhas high 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 HPP1 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. Studies have found that hypermethylation of hyperplastic polyposis protein 1 (HPP1, GeneID: 23671) is involved in the occurrence and development of various malignant tumors, including gastric adenocarcinoma (Ivanauskas A, et al. Dig Liver Dis. 2008Dec; 40(12): 920-6.), thymoma (Hirose Y, et al.Lung Cancer.2009May; 64(2):155-9), hepatocellular carcinoma (Vivekanandan P, et al.Mod Pathol.2008Jun; 21(6): 670-5), colon cancer (Wallne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 李兆申王小玮高军杜奕奇龚燕芳
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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