DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法

doi:10.16288/j.yczz.15-129

遗传 ›› 2015, Vol. 37 ›› Issue (9): 939-944.doi: 10.16288/j.yczz.15-129

DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法

刘洋洋¹, 崔恒宓^{1, 2}

1. 南京大学医学院,江苏分子医学重点实验室,南京 210093;
2. 扬州大学表观遗传学及表观基因组学研究所,扬州 225009

收稿日期:2015-03-26 修回日期:2015-06-16 出版日期:2015-09-20 发布日期:2015-09-20
通讯作者: 崔恒宓,博士,教授,研究方向：表观遗传学。E-mail: hcui@nju.edu.cn
作者简介:刘洋洋,硕士研究生,专业方向：表观遗传学。E-mail: yangyang01234@126.com
基金资助:
国家自然科学基金项目(编号：81171965,81372237)和国家重点基础研究发展计划项目(937计划)(编号：2012CB517605)资助

The method of estimating bisulfite conversion rate in DNA methylation analysis

Yangyang Liu¹, Hengmi Cui^{1, 2}

1. Jiangsu Province Key Lab of Molecular Medicine, School of Medicine, Nanjing University, Nanjing 210093, China;
2. Institute of Epigenetics & Epigenomics, Yangzhou University, Yangzhou 225009, China

Received:2015-03-26 Revised:2015-06-16 Online:2015-09-20 Published:2015-09-20

摘要/Abstract

摘要： 为建立一种评估重亚硫酸盐处理DNA样本后胞嘧啶转化效率的有效方法,以两组不同的TaqMan qPCR检测梯度稀释的重亚硫酸盐处理和未处理的DNA标准品,建立转化与未转化的DNA Ct值以及对应的DNA拷贝数的标准曲线。使用相同的探针定量检测重亚硫酸盐处理后的DNA样本评估转化效率。结果显示该方法应用两组探针,根据相应的标准曲线,精确评估样本经重亚硫酸盐处理的转化效率。使用已知转化和未转化拷贝数的混合DNA作为模板,证实了该方法的可靠性。同时也对不同重亚硫酸盐试剂盒处理DNA的转化效率进行了评估,结果显示,该方法能够有效地评估DNA样品重亚硫酸盐的转化效率,为DNA甲基化准确分析提供了可靠快捷的方法。

关键词: DNA甲基化, 重亚硫酸盐, TaqMan qPCR, 转化率

Abstract: To establish an effective method to estimate the conversion rate of bisulfite-treated genomic DNA, TaqMan qPCR assay was performed using probes and primers that are specific for bisulfite-converted or -unconverted DNA standard samples separately. Then two linear standard curves were generated by plotting Ct values against logarithm of absolute DNA amount with serial dilutions of the bisulfite-converted or unconverted DNA samples. Based on two standard curves, the unknown bisulfite-treated genomic DNA sample was analyzed using the same TaqMan probes and the bisulfite conversion rate was precisely estimated. This method was further verified to be reliable using known mixed bisulfite-converted and -unconverted DNA templates as well as DNA samples treated with different bisulfite kits. These results showed that this method can effectively estimate bisulfite conversion rate of genomic DNA and thus provides a reliable and quick method for accurate analyses of DNA methylation.

Key words: DNA methylation, bisulfite, TaqMan qPCR, conversion rate

刘洋洋, 崔恒宓. DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法[J]. 遗传, 2015, 37(9): 939-944.

Yangyang Liu, Hengmi Cui. The method of estimating bisulfite conversion rate in DNA methylation analysis[J]. HEREDITAS(Beijing), 2015, 37(9): 939-944.

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DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法

The method of estimating bisulfite conversion rate in DNA methylation analysis

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