一种用于DNA甲基化分析的改进型亚硫酸氢盐转化法  

An Improved Agarose Bisulfite Method for DNA Methylation Analysis

作　　者：赵贵森[1] 郑海燕[2] 莫耀南[2] 冯文芳[1] 杨渝珍[1] 

机构地区：[1]华中科技大学同济医学院生物化学与分子生物学系,武汉430030 [2]河南科技大学法医学院,洛阳471003

出　　处：《中国生物化学与分子生物学报》2007年第10期876-880,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30500085)~~

摘　　要：DNA甲基化分析是认识生理、病理条件下基因表达变化的重要途径.亚硫酸氢盐转化是DNA甲基化分析的瓶颈.本文旨在改进琼脂糖-亚硫酸氢盐DNA处理方案(agarose bisulfite method),建立一种简便稳定、适合常规甲基化分析的亚硫酸氢盐转化法.把DNA包入普通琼脂糖,以饱和亚硫酸氢盐在较高的温度下快速处理,然后用离心柱型琼脂糖凝胶DNA回收试剂盒,集DNA凝胶回收、脱盐、脱磺基和纯化于一体,完成整个转化过程.Bisulfite-PCR、克隆测序和酶切法分析转化率、转化特异性和转化物的质量.用该方案处理的HeLa细胞DNA,多个片段的转化率均大于98%,甲基化片段96.2%的CpG保持不变,可以扩增605bp的较大片段,灵敏度介于普通法和琼脂糖亚硫酸氢盐法之间,而重复性较二者都好.改良后的方案简化了操作流程,快速稳定,易学易用,可实现高效特异转化,适合于一般实验者对常规检材进行DNA甲基化分析.DNA methylation analysis is an important pathway to understand gene expression changes under physiological or pathological conditions. Bisulfite conversion is the bottleneck of DNA methylation analysis. To improve the bisulfite modification technique in DNA methylation analysis, an agarose embedding based bisulfite conversion procedure was developed. In this simplified procedure, genomic DNA embedded into agarose was treated with concentrated bisulfite at an elevated temperature to accelerate the conversion speed; then, all subsequent steps （gel extraction, desalting, desulfonation and the final purification） were included in a modified gel extraction procedure. Using this method, HeLa DNA was modified and assayed by bisulfite genomic sequencing （BSG） and combined bisulfite restriction analysis （COBRA）. The conversion rate was more than 98 %, conversion specificity was more than 96.2 % and of the converted DNA was confirmed with good quality and validity. The modification improved the original protocol in technical simplicity, time saving and more suitable for downstream applications. These, combined with its high reproducibility, would make it a valuable tool in DNA methylation studies.

关 键 词：DNA甲基化 亚硫酸氢钠 琼脂糖 DNA凝胶回收 

分 类 号：Q75[生物学—分子生物学]