Quantitative detection method of HPP1 gene methylation

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...

AI Technical Summary

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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