一种用于检测DNA甲基化的改良亚硫酸氢盐转化方法。
A modified bisulfite conversion method for the detection of DNA methylation.
作者信息
Wang Hanze, Ke Huican, Zheng Yansong, Lai Junzhong, Luo Qianping, Chen Qi
机构信息
Fujian Key Laboratory of Innate Immune Biology, Biomedical Research Center of South China, College of Life Science, Fujian Normal University Qishan Campus, 1 Keji Road, College Town, Fuzhou, Fujian 350117, China.
Department of Hepatobiliary Pancreatic Surgery, The First Affiliated Hospital of Fujian Medical University, Fujian, China.
出版信息
Epigenomics. 2017 Jul;9(7):955-969. doi: 10.2217/epi-2016-0174. Epub 2017 May 26.
AIM
Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA.
METHODS
Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing.
RESULTS
We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits.
CONCLUSION
Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.
目的
我们的目的是改进用于检测DNA中5-甲基胞嘧啶的亚硫酸氢盐转化常规程序。
方法
通过甲基化敏感熔解曲线分析评估不同亚硫酸氢盐、亚硫酸氢盐转化温度、抗氧化剂和变性剂对DNA转化和降解的影响。在不同基因上测试改良方法，并通过亚硫酸氢盐测序分析转化效率。
结果
我们开发了一种改良的亚硫酸氢盐转化方法，该方法可在2小时内完成此过程。我们证明高温变性是DNA降解的主要原因，添加乙二醇二甲醚是加速亚硫酸氢盐转化的有效方法。转化效率与许多其他商业试剂盒相当。
结论
我们改良的亚硫酸氢盐转化方法简单、成本效益高且耗时少，并且与不同基因和样品兼容，因此在未来研究和临床应用中具有巨大潜力。
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