A systematic evaluation of whole genome amplification of bisulfite-modified DNA.
A systematic evaluation of whole genome amplification of bisulfite-modified DNA.
复制标题
对硫酸含石修饰DNA的整个基因组扩增的系统评估。
DOI：
10.1186/1868-7083-4-22
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发表时间：
2012-11-22
影响因子：
5.7
通讯作者：
Iwamoto K
中科院分区：
文献类型：
--
作者：
Bundo M;Sunaga F;Ueda J;Kasai K;Kato T;Iwamoto K
详细研究DNA甲基化谱应该是表观遗传学研究的第一步。虽然亚硫酸氢钠修饰基因组DNA是DNA甲基化分析的金标准方法，但这种方法会导致大部分DNA材料的损失。亚硫酸氢盐修饰DNA的全基因组扩增（WGA）有望提供丰富的材料来源，但其有效性尚未得到彻底评估。在这项研究中，我们评估了偏置扩增的程度，在WGA的亚硫酸氢盐修饰的DNA和独立的WGA反应的再现性。我们分别进行了三次基于多重位移放大的WGA。每个实验包括使用10或50 ng亚硫酸氢盐修饰的DNA作为模板的两个反应。在约450，000个CpG位点处比较WGA产物与原始亚硫酸氢盐修饰的DNA之间的DNA甲基化水平。使用足够量的亚硫酸氢盐修饰的DNA用于WGA对于下游应用至关重要。在DNA甲基化水平的中间范围内发现了与原始亚硫酸氢盐修饰的DNA的相当大的偏差。高甲基化和低甲基化的分布是相等的，这表明每个CpG位点的偏离是随机发生的。平均化来自独立放大的WGA产品的数据显著提高了整体质量。亚硫酸氢盐修饰的DNA的WGA可能是表观遗传学研究的一个有价值的工具，但需要仔细的实验设计和数据解释。
Studying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA) of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites. Using a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality. WGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.
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影响因子：
46.9
作者：
Ball, Madeleine P.;Li, Jin Billy;Gao, Yuan;Lee, Je-Hyuk;LeProust, Emily M.;Park, In-Hyun;Xie, Bin;Daley, George Q.;Church, George M.
通讯作者：
Church, George M.
影响因子：
3.7
作者：
Vaissiere, Thomas;Cuenin, Cyrille;Herceg, Zdenko
通讯作者：
Herceg, Zdenko
影响因子：
16.8
作者：
Lorincz, MC;Dickerson, DR;Groudine, M
通讯作者：
Groudine, M
影响因子：
7
作者：
Hosono, S;Faruqi, AF;Lasken, RS
通讯作者：
Lasken, RS
影响因子：
9.8
作者：
Flanagan, James M.;Popendikyte, Violeta;Petronis, Arturas
通讯作者：
Petronis, Arturas