一种快速定量检测p16基因甲基化方法的建立  

Chemiluminescence Quantitative Detection of pl6 Promoter Hypermethylation Based on Mismatch Hybridization

作　　者：姚群峰[1] 郝巧玲[1] 邹立君[1] 张利平[1] 徐顺清[1] 周宜开[1] 

机构地区：[1]华中科技大学同济医学院公共卫生学院环境医学研究所,武汉市430030

出　　处：《医学分子生物学杂志》2005年第5期351-354,共4页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.39990570)~~

摘　　要：目的建立一种基于错配杂交-化学发光检测的p16基因启动子区过甲基化的定量分析方法。方法用亚硫酸氢钠修饰基因组DNA,所有未甲基化的胞嘧啶都被转变为尿嘧啶,而甲基化的胞嘧啶则不发生变化。设计合成一对不含CpG位点的引物同时扩增甲基化或非甲基化目的DNA片段,用两根分别与甲基化及非甲基化CpG位点互补的寡核苷酸探针与扩增产物进行杂交,化学发光检测,通过两根探针的杂交信号强度之比确定样品DNA中甲基化的p16基因的比例。结果检测结果与肿瘤细胞DNA样品中甲基化的p16基因的量成正比,而且与其表达水平呈逆相关。结论与现有方法相比,本法是一种检测快速、操作简便的p16基因甲基化的定量检测方法。Objectives To establish a novel method for quantitative detection of p16 promoter hypermethylation based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite, thereby converting all unmethylated, but not methylated, cytosines to uracil. A pair of primer which had no CpG sites was designed for amplifying target DNA containing methylated or unmethylated CpG sites. Two oligonucleotide probes, which perfectly matched the methylated and unmethylated CpG sequences respectively, were synthesized. The PCR products spanning CpG sites were hybridized with two oligonucleotide probes and the hybrids were detected by chemiluminescence. Results The percentage of methylated target sequences can be estimated by calculating the ratio of signals obtained with two probes. Conclusion Compared with the existing methods, this assay is simple, sensitive, and specific for quantitative analysis of the promoter methylation of p16 gene.

关 键 词：P16基因 甲基化 化学发光 错配杂交 

分 类 号：R349.8[医药卫生—基础医学]