Department of Pathology, Harvard Medical School and Immune Disease Institute, Boston, Massachusetts, United States of America.
PLoS One. 2010 Jan 26;5(1):e8888. doi: 10.1371/journal.pone.0008888.
We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing.
METHODOLOGY/PRINCIPAL FINDINGS: We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1-2 nucleotides.
We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.
我们最近发现 TET 家族的酶可将 DNA 中的 5-甲基胞嘧啶（5-mC）转化为 5-羟甲基胞嘧啶（5-hmC）。5-hmC 在胚胎干细胞和浦肯野神经元中大量存在。胞嘧啶的甲基化状态通常通过与亚硫酸氢钠反应，随后进行 PCR 扩增来评估。与亚硫酸氢钠反应会促进胞嘧啶脱氨，而 5-甲基胞嘧啶（5-mC）与亚硫酸氢盐反应不佳，且不易脱氨。由于 5-hmC 与亚硫酸氢盐反应生成胞嘧啶 5-亚磺酸盐（CMS），我们想知道含有 5-hmC 的 DNA 在亚硫酸氢盐测序中的行为。
方法/主要发现：我们使用具有不同胞嘧啶分布的合成寡核苷酸作为模板，生成含有 C、5-mC 和 5-hmC 的 DNA。这些生成的 DNA 同时进行亚硫酸氢盐处理，然后暴露于促进胞嘧啶脱氨的条件下。5-hmC 转化为 CMS 的程度估计为 99.7%。对 PCR 产物进行测序表明，5-mC 和 5-hmC 在后处理均不会发生 C 到 T 的转换，这证实了这两种修饰的胞嘧啶在亚硫酸氢盐技术中无法区分。CMS 构成所有碱基（28/201）的大部分的 DNA 的扩增效率远低于那些碱基为 5-mC 或尿嘧啶（后者由胞嘧啶脱氨产生）的 DNA。通过一系列引物延伸实验，我们发现 Taq 聚合酶在 CMS 修饰位点停滞，特别是当两个 CMS 碱基彼此相邻或间隔 1-2 个核苷酸时，CMS 含有 DNA 的扩增效率降低。
我们已经证实，广泛使用的亚硫酸氢盐测序技术无法区分 5-mC 和 5-hmC。此外，我们表明，CMS 是 5-hmC 经亚硫酸氢盐转化的产物，在 PCR 过程中往往会使 DNA 聚合酶停滞，这表明 DNA 中高度羟甲基化的区域可能在定量甲基化分析中代表性不足。
