The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.
The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.
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DOI：
10.1371/journal.pone.0008888
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发表时间：
2010-01-26
期刊：
影响因子：
3.7
通讯作者：
Rao A
中科院分区：
文献类型：
--
作者：
Huang Y;Pastor WA;Shen Y;Tahiliani M;Liu DR;Rao A
我们最近发现TET家族的酶在DNA中将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含量很高。胞嘧啶的甲基化状态通常通过与亚硫酸氢钠的反应进行评估，随后进行PCR扩增。与亚硫酸氢钠反应促进胞嘧啶脱氨，而5-甲基胞嘧啶（5-mC）与亚硫酸氢钠反应较差，不易脱氨。由于5-hmC与亚硫酸氢盐反应生成胞嘧啶5-亚甲基磺酸（CMS），我们询问含有5-hmC的DNA在亚硫酸氢盐测序中的表现。我们使用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。得到的dna平行于亚硫酸氢盐处理，随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%。PCR产物测序显示，亚硫酸氢盐处理后，5-mC和5-hmC均未发生C-to-T转变，证实亚硫酸氢盐技术无法区分这两种修饰的胞嘧啶。CMS占所有碱基大部分的DNA（28/201）比这些碱基为5-mC或尿嘧啶（后者由胞嘧啶脱氨产生）的DNA扩增效率低得多。通过一系列引物延伸实验，我们发现含有CMS的DNA的低效扩增是由于CMS修饰位点上Taq聚合酶的停滞，特别是当两个CMS碱基相邻或相隔1-2个核苷酸时。我们已经证实，广泛使用的亚硫酸盐测序技术不能区分5-mC和5-hmC。此外，我们发现亚硫酸氢盐转化5-hmC的产物CMS在PCR过程中往往会使DNA聚合酶停滞，这表明DNA的密集羟甲基化区域在定量甲基化分析中可能代表性不足。
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. 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.
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