死亡相关蛋白激酶基因启动子甲基化特异性PCR法的建立与初步应用

Establishment and preliminary applification of methylation-specific PCR for detecting methylation of death-associated protein kinase promoter

目的:建立甲基化特异性PCR(MSP)技术检测死亡相关蛋白激酶(DAPK)基因启动子甲基化。方法:设计针对DAPK基因启动子的MSP引物,建立MSP检测体系对甲基化阳性模板进行检测,评价其特异性、重复性和灵敏性,并经测序鉴定。结果:所建立的甲基化MSP方法仅能扩增出甲基化阳性模板,不能扩增出未甲基化阳性模板和未硫化处理的DNA模板;对不同梯度稀释的甲基化阳性模板进行检测,其最大敏感性可达2%;在不同时间进行甲基化MSP检测的结果均一致。结论:成功建立的DAPK基因启动子甲基化MSP检测方法具有良好的特异性、灵敏性和重复性,可用于肿瘤标本的批量检测。

Objective： To establish the methylation-specific PCR（MSP） for the detection of promoter methylation of death-associated protein kinase（DAPK） gene.Methods： The primers of MSP aiming at the DAPK promoter were designed.MSP was established to detect the methylation positive DNA templates.The specificity,sensibility and repeatability were evaluated. Results： The established methylated MSP could only amplify the methylation positive DNA template,but not the unmethylated DNA template and unmodified DNA template.In a series of dilution,methylated MSP could detect 2% methylation positive DNA templates.The amplified results of same sample were identical on different time. Conclusion： The methylated MSP technique for detecting of DAPK methylation was established successfully and can be used for analyzing samples from cancer patients.