巢式甲基化特异性聚合酶链反应在p16基因启动子过甲基化检测中的应用

Detection of p16 promoter hypermethylation by nested methylation specific polymerase chain reaction

目的 介绍巢式甲基化特异性聚合酶链反应 (nMSP)法 ,探讨了最佳PCR扩增条件 ,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 ,将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特异引物 ,进行巢式聚合酶链反应扩增 ,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基因启动子甲基化。用甲基化特异性聚合酶链反应法 (MSP)和nMSP法分别检测 34例非小细胞肺癌 (NSCLC)患者血清 ,p16基因启动子甲基化阳性率分别为 5 3% (18/34)和 74 % (2 5 /34) ,nMSP法具有更高的灵敏度。结论 筑巢式甲基化特异性聚合酶链反应是一种灵敏度高、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 A sensitive and specific method PCR-based for rapid analysis of the promoter methylation status, Nested-MSP, was introduced, and the method was used detect p16 promoter hypermethylation status in fresh tumor tissue、paraffin-embedded tissue and serum sample. Methods Target DNA was denatured by NaOH, then single strands DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequent a nested amplification with primers specific for methylated versus unmethylated DNA. PCR product was detected by gel electrophoresis. Results The promoter regions methylation of p16 gene was found in the fresh tumor tissues, paraffin-embedded tissue, and serum of primary non-small cell lung cancer(NSCLC) patients. Aberrant promoter rmethylation of the p16 gene was detected in 18 of 34 (53%) serum samples by MSP and in 25 of 34 (74%) by nMSP. Conclusion Nested methylation specific polymerase chain reaction (nMSP) is a simple, sensitive and specific method for rapid analysis of the promoter methylation status of many genes.