DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法.pdfVIP

Hereditas (Beijing) 2015 年9 月, 37(9): 939 ―944 技术与方法 DNA 甲基化分析中重亚硫酸盐处理DNA 转化效率的 评估方法 1 1,2 刘洋洋 ，崔恒宓 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 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。 DNA 甲基化；重亚硫酸盐；TaqMan qPCR；转化率 关键词: The method of estimating bisulfite conversion rate in DNA methylation analysis 1 1,2 Yangyang Liu , Hengmi Cui 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 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 loga- rithm 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 differ- ent bisulfite kits. These results showed that this method can effectively esti