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  

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

作　　者：徐新娟[1] 韩钰[2] 丁文柏[1] 盛德乔[3] 陈世雄[1] 曾凡军[1] 高宝安[1] 夏道奎[1] 胡旭[1] 张春芳[1] 

机构地区：[1]三峡大学第一临床医学院,湖北宜昌443003 [2]三峡大学分子生物学研究所,湖北宜昌443002 [3]三峡大学医学院生物化学教研室,湖北宜昌443002

出　　处：《现代生物医学进展》2008年第9期1671-1673,1633,共4页Progress in Modern Biomedicine

基　　金：三峡大学博士研究启动基金(0620060087)

摘　　要：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。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 fiberoptie bronehoscopy （FB）, Methods： Target DNA was denatured by NaOH, then single strand DNA was modified by sodium bisulfite. All unmethylated, but not methylated cytosines were convetted to uraeils, 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,

关 键 词：巢式甲基化特异性PCR(nMSP) WIF-1 基因 甲基化 

分 类 号：R734.2[医药卫生—肿瘤] R394.2[医药卫生—临床医学]