利用甲基化特异性引物高通量检测DNA甲基化

High Throughput Quantification of Genomic DNA Methylation via Methylation-specific Primers

建立一种基于甲基化特异性引物和SAGE技术的高通量DNA甲基化定量检测新方法(MSP-SAGE),首先利用亚硫酸氢钠对基因组DNA进行处理,使未甲基化的C转变为U,而甲基化的CpG不变.将处理和未处理的DNA双链变性后用随机引物PNNNNCG对存在含有CG的单链进行延伸,而无甲基化CG的单链处则不能延伸;将差异延伸的单链序列和频次信息经过系列分子操作后,引入PCR扩增模板;对中间带有未知序列的PCR扩增产物进行串连克隆测序.将来自于未处理组和处理组的某一CpG位点的序列出现的次数定义为[Tags]A和[Tags]B,将标准系列的实际甲基化水平和[Tags]B/[Tags]A之间建立线性回归方程.根据每一CpG位点的[Tags]B/[Tags]A比值可反推该位点的甲基化水平.MSP-SAGE具有良好的线性,基于标准系列的[Tags]B/[Tags]A与其实际甲基化水平的标准曲线方程为y=1.455x(R2=0.984,P<0.01).MSP-SAGE的回收率在95%到110%之间,精确度位于4.2%和10.5%,检测限在3%左右,单次检测通量可达24个CpG位点.MSP-SAGE是一种很有应用前途的高通量DNA甲基化定量检测方法.

To test a novel method based on methylation-specific primers and SAGE（MSP-SAGE） for high-throughput quantification of genomic DNA methylation,a six-mer methylation-specific primer was used to extend methylated CpG sequences after bisulfite treatment,the obtained sequences were then subjected to PCR amplifications.Digestion of MmeⅠ,the 17bp tags of methylated CpG sequences were concatenated for SAGE analyses and cloned for sequencing,which were used to quantify the genomic methylation status and determine the locations of methylated alleles.The linear calibration equation of our MSP-SAGE assay was y=1.455x（R^2=0.984,P〈0.01）,which was capable of measuring the degree of genomic methylation ranging from 5% to 100% with the precision of 4.2%—10.5%.Up to 24 alleles could be quantified simultaneously and the detection limit was approximately 3%.The methylation patterns determined by MSP-SAGE were validated by bisulfite sequencing.Our study has demonstrated that MSP-SAGE was an accurate and reliable high throughput assay for the analysis and quantification of DNA methylation.