巢式PCR法检测不同类型标本中p16基因启动子过甲基化

Detection of Promoter Hypermethylation of p16 Gene in Different Kinds of Samples by Nested Methylation Specific Polymerase Chain Reaction

目的 介绍一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),探讨该方法最佳应用条件,并用该法分析新鲜癌组织、石蜡包埋组织及肿瘤患者血清中p16基因启动子的甲基化状态。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种类型的标本中都检测出了p16基因启动子的甲基化,比普通的甲基化特异性PCR法具有更高的灵敏度。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分析。

Objective To introduce a sensitive and specific method PCR-based for rapid analysis of the promoter methylation status, nested mythylation specific polymrase chain reaction (nMSP), explore the optimal conditions for this method and detect p16 promoter hypermethylation status in fresh tumor tissue, paraffin-embedded tissue and serum sample by this method. Methods Target DNA was denatured by NaoH, then single strands DNA was modified by sodium bisulfite. All unmethylated, but not methylated cytosines were converted to uracil. 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 methylation of p16 gene was detected in the fresh tumor tissues, paraffin-embedded tissue, and serum of the patients with primary non-small cell lung cancer. Conclusion nMSP is a simple, sensitive and specific method for rapid analysis of the promoter methylation status of many genes.