巢式甲基化特异性PCR检测不同类型组织标本中WIF-1基因启动子异常甲基化

目的:采用一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),检测外科手术切除新鲜组织、石蜡包埋组织及纤维支气管镜活检组织中WIF-1基因启动子的异常甲基化状态。方法:将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式PCR扩增,最后经凝胶电泳检测目的片段。结果:在3种类型的原发性非小细胞肺癌标本中都检测出了WIF-1基因启动子的异常甲基化。结论:巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化的分析。

Detection of Promoter Abnormal Methylation of WIF-1 Gene in Different Kinds of Samples by Nested Methylation Specific Polymerase Chain Reaction

Objective:To use nested-methylation specific polymerase chain reaction(nMSP), a sensitive and specific method PCR-based for rapid analysis of the promoter methylation status, to detect WIF-1 gene promoter abnormal methylation status in surgical resected fresh tumor tissue, paraffin-embedded tissue and biopsy samples of fiberoptic bronchoscopy(FB).Methods:Target DNA was denatured by NaOH, then single strand DNA was modified by sodium bisulfite.All unmethylated, but not methylated cytosines were con-verted to uracils.The primers specific for methylated versus unmethylated DNA were designed and amplified by nested-PCR.The target fragment was verified by gel electrophoresis.Results:The promoter abnormal methylation of WIF-1 gene was detected in all the three types of primary non-small cell lung cancer tissues.Conclusions:nMSP was a simple, sensitive and specific method for rapid analysis of the promoter abnormal methylation status of many genes.