A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements -: art. no. e38
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements -: art. no. e38
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DOI：
10.1093/nar/gnh032
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Issa, JPJ
中科院分区：
文献类型：
--
作者：
Yang, AS;Estécio, MRH;Issa, JPJ
我们报道了一种研究DNA甲基化的方法，该方法基于使用亚硫酸氢盐处理DNA和同时PCR多个DNA重复元件，如Alu元件和长散布核苷酸元件（LINE）。PCR产物代表约15000个基因组位点的库，可用于直接测序、选择性限制性消化或焦磷酸测序，以定量DNA甲基化。通过限制性消化或焦磷酸测序，测定是可重复的，测定之间的标准偏差仅为2%。使用这种方法，我们发现Alu元件中几乎三分之二的CpG甲基化位点发生突变，但在剩余的甲基化靶位点中，87%被甲基化。由于重复元件的高度甲基化，该测定在检测DNA甲基化的降低中特别有用，并且该测定通过检查用甲基化抑制剂5-氮杂-2 '脱氧胞苷（DAC）处理的细胞系来验证，其中我们发现在处理的3天内Alu元件降低1-16%，LINE甲基化降低18-60%。该方法可用作全基因组甲基化变化的替代标记。此外，与以前评估整体DNA甲基化的方法相比，它的劳动强度更低，需要的DNA更少。
We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.