基于错配杂交化学发光定量检测MGMT基因过甲基化

A Novel Method for Quantitative Detection of MGMT Promoter Hypermethylation Based on Mismatch Hybridization and Chemiluminescence

目的利用错配杂交和化学发光技术,建立一种检测MGMT基因启动子区过甲基化的定量分析方法。方法基因组DNA经过亚硫酸氢钠修饰,所有未甲基化的胞嘧啶都被转变为尿嘧啶,而甲基化的胞嘧啶则不发生变化。设计特异的PCR引物(不含CpG位点),同时扩增含有CpG位点甲基化或非甲基化目的 MGMT基因启动子区,用两条分别与甲基化及非甲基化CpG位点互补的寡核苷酸探针与扩增产物杂交,化学发光检测,通过两条探针的杂交信号强度之比确定样品DNA中MGMT甲基化的程度。结果检测混合样品中MGMT基因的甲基化水平与结果完全相符,并可用于肿瘤组织样品的MGMT甲基化定量检测。结论与现有方法相比,本法是一种检测快速、操作简便的MGMT基因甲基化的定量检测方法。

Objective To establish a novel method for quantitative detection of MGMT promoter hypermethylation based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite,converting all unmethylated,but not meth- ylated,cytosines to uracil, and subsequent a pair of primer which have no CpG sites was designed for amplification target DNA contained meth- ylated or unmethylated CpG sites. Two oligonucleotide probes ,which perfectly match the methylated and unmethylated CpG sequences respec- tively,were synthesized. The PCR products spanning CpG sites were hybridized with two oligonucleotide probes and the hybrids were detected by chemiluminescence. Results The methylation level of MGMT gene by using the method of detection of mixed samples and tumor tissues, and the results are fully consistent with the actual. Conclusion Campared with the existing methods, this asssy is a simple, sensitive , and specific method for quantitative analysis of the promoter methylation of MGMT gene.