Taqman hydrolysis probe and method for quantitatively detecting methylation level of MGMT (O6-methylguanine-DNA-methyltransferase)

Abstract

The invention provides a Taqman fluorescent hydrolysis probe and a method for quantitatively detecting the methylation level of MGMT (O6-methylguanine-DNA-methyltransferase) by using the Taqman hydrolysis probe. The fundamental sequence of the Taqman fluorescent hydrolysis probe is GATTTGGTGAGTGTUTGGG, and the complementary region of the fundamental sequence and a genomic DNA (deoxyribonucleic acid) sequence extends upstream no more than 5nt and extends downstream no more than 5nt. According to the invention, the specificity of PCR (polymerase chain reaction) is doubly guaranteed; the percent of a methylated DNA template in a mixed sample can be quantitatively calculated; and the method has all the advantages (such as speediness, sensitiveness, no pollution, and the like) of the real-time quantitative PCR. The application of detection results of the invention can provide clinical guide for medicaments for carrying out chemotherapy on malignant tumors such as cerebral gliomas and the like; and the method has the advantages of high specificity, high sensitivity, accurate quantification, and the like.

Description

technical field [0001] The invention relates to a Taqman hydrolysis probe and a method for detecting the methylation degree of MGMT. Background technique [0002] The human MGMT gene is located on chromosome 10q26, with a total length of about 170kb, consisting of 5 exons and 4 introns. MGMT protein can catalyze guanine O in DNA molecules without any cofactor or other proteins 6 The alkyl group at the position is transferred to the cysteine ​​residue at position 145 of MGMT itself, the guanine can be restored, and the structure and function of DNA can be restored, thus protecting cells from the damage of alkylating agents. The cytotoxicity of anticancer drugs containing methyl and chloroethyl mainly depends on the ability to form alkyl groups between them and DNA to cause intra-chain cross-linking. However, MGMT can remove the alkyl group from the alkylated DNA strand, making treatment with alkylating agents ineffective. Therefore, MGMT can eliminate the carcinogenic effe...

AI Technical Summary

Problems solved by technology

This is a classic methylation research method. Its advantages are: relatively simple, low cost, clear methylation sites, and easy interpretation of experimental results. The disadvantages are: a. Since CG is not limited to CCGG sequences, it is not The CG in this sequence will be ignored; b. Only when the methylation status of key sites related to transcription is detected, the results of this detection method are meaningful; c. Relatively speaking, the Southern method is more complicated and requires The amount of sample is large; d, there is a problem of false positives caused by incomplete enzyme digestion; d, 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 regions, which limits MSP usage of

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