p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关，但p16基因缺失率较低，突变更为罕见，p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点，现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、

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

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.