p16基因甲基化的芯片定量检测  

A Methylation-specific Oligonuceotide Microarray for Quantitative Analysis of p16 Gene

作　　者：沈佳尧[1] 侯鹏[2] 祭美菊[2] 李松[2] 何农跃[2] 

机构地区：[1]苏州大学生命科学学院,苏州215007 [2]东南大学生物科学与医学工程系,吴健雄实验室,南京210096

出　　处：《高等学校化学学报》2005年第3期449-451,共3页Chemical Journal of Chinese Universities

基　　金：国家"九七三"计划 (批准号 :G19980 5 12 0 4);江苏省高新技术项目 (批准号 :BG2 0 0 10 10 );国家自然科学基金 (批准号 :60 0 710 0 1)资助 .

摘　　要：To quantitatively detect hypermethylation and to analyze methylation pattern, a methylation-specific oligonucleotide microarray for quantitative analysis was developed. The method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample was hybridized to a set of oligonucleotide arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions,and quantitative differences in hybridization were determined by fluorescence analysis. Four clinical samples of gastric cancer were quantitatively detected and methylation pattern of five methylated clones were analyzed with the methylation-specific oligonucleotide microarray. The results of microarray hybridization were in agreement with bisulfite genomic DNA sequencing. It showed that methylation-specific oligonucleotide microarray for quantitative analysis is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.To quantitatively detect hypermethylation and to analyze methylation pattern, a methylation-specific oligonucleotide microarray for quantitative analysis was developed. The method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample was hybridized to a set of oligonucleotide arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions,and quantitative differences in hybridization were determined by fluorescence analysis. Four clinical samples of gastric cancer were quantitatively detected and methylation pattern of five methylated clones were analyzed with the methylation-specific oligonucleotide microarray. The results of microarray hybridization were in agreement with bisulfite genomic DNA sequencing. It showed that methylation-specific oligonucleotide microarray for quantitative analysis is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.

关 键 词：DNA甲基化 荧光强度标准曲线 p16基因甲基化检测型基因芯片 

分 类 号：O657[理学—分析化学]