定量检测E-cadherin基因启动子区甲基化芯片的建立

目的：拟建立一种能够定量检测抑癌基因E-cadherin甲基化改变的基因芯片.方法：用亚硫酸氢盐处理基因组DNA,并以此为模板进行PCR扩增.目的序列中未甲基化的CpG住点被翻转成TpG,而甲基化的CpG位点保持不变.设计5组探针构建一种检测E-cadherin基因启动子区17个CpG住点甲基化改变的芯片,每组探针包括一对非甲基化探针和甲基化探针,检测相邻的3或4个CpG位点.结果：绘制标准曲线显示,芯片上探针的可重复性和精确性很好,并定量检测了1例白血病样本的甲基化改变.结论：基因芯片能够作为一种定量检测基因多个CpG位点甲基化改变的有效工具,并用于肿瘤的研究.

Establishment of microarray-based method for quantificationally detecting methylation changes of E-cadherin gene promoter

Objective To establish a microarray-based method for quantificationally detecting methylation changes of a tumor suppressor gene E-cadherin.Methods This 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.Five sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect 17 CpG sites in the promoter of E-cadherin gene,each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 or 4 CpG sites in close proximity.Results By drawing a standard curve we found the accuracy and reproducibility of the probes designed for microarray hybridization was very good.This standard curve was used to calibrate the levels of methylation changes detected in a leukemia sample by microarray assay.Conclusion Microarray assay can be applied as a useful tool for mapping methylation changes in multiple CpG loci and for researching cancer.