Methylation quantitative detection method of PCDH8 gene

Abstract

The invention belongs to the technical field of biomedicine. Based on the fact that the abnormal methylation of genes are involved in the generation and development of tumors, the invention aims to overcome the defects of a methylation sensitive restriction enzyme method, a bisulfite sequencing method and a methylation specificity PCR (Polymerase Chain Reaction) method, and provide a methylation quantitative detection method of the PCDH8 (Protocadherin-8) gene, with simple and convenient operation and high sensitivity. The detection method comprises the steps of: taking pancreatic cancer tissues, extracting DNA to prepare a standard curve sample of ACTB (Actin, Beta), and preparing a standard curve sample of PCDH8; extracting the DNA of a tissue to be detected, carrying out chemical decoration, designing a methylation primer and a probe for a CPG island in a human PCDH8 gene promoter area, and designing a BSP primer and a probe for a CPG island in a human reference gene ACTB promoter area, carrying out real-time quantitative PCR on the DNA subjected to the sulfite treatment by using a Taqman-MGB real-time quantitative PCR method, and calculating out a quantitative value on the methylation degree of a PCDH8 gene. The method has the advantages of rapidness, correctness and 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 PCDH8 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. The study found that proto-cadherin (PCDH8, GeneID: 5100) is a candidate tumor suppressor gene of breast cancer, and the loss of PCDH8 can promote the formation of epithelial cancer by destroying the information transmission between cells, which is beneficial to tissue Organ and suppress carcinogenesis (Yu JS, et al.PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer. Oncogene.2008 Aug; 27(34):4657-...

AI Technical Summary

Problems solved by technology

[0005] (1) Since CG is not limited to CCGG sequence, CG not in this sequence will be ignored;

[0006] (2) Only when the methylation status of key sites related to transcription is detected, the results of this detection method are meaningful; (3) Relatively speaking, the Southern method is more complicated and requires a large amount of samples; (4) ) has the problem of false positives caused by incomplete enzyme digestion; (5) is not suitable for mixed samples

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.

[0009] At present, there is no quantitative detection method related to PCDH8 gene methylation

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