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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 a highly sensitive method for DNA methylation analysis, namely nested-MSP (nMSP), detection of surgical excision of fresh tissue, paraffin-embedded tissue and fibroscopic bronchoscopic biopsy The abnormal methylation status of WIF-1 gene promoter. Methods: Genomic DNA was denatured to single-stranded, bisulphite-modified single-stranded DNA, all unmethylated cytosines were converted to uracil, while methylated cytosines were unchanged. Specific primers for methylated and unmethylated alleles were designed and amplified by nested PCR. Finally, the target fragments were detected by gel electrophoresis. RESULTS: Aberrant methylation of the WIF-1 gene promoter was detected in all three types of primary non-small cell lung cancer specimens. Conclusion: Nested methylation-specific PCR is a highly sensitive and specific methylation detection method, which can be widely used in the methylation analysis of promoter of different types of specimens.