Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants
Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants
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DOI：
10.1016/j.bmc.2016.07.016
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发表时间：
2016-09-15
影响因子：
3.5
通讯作者：
Suetake, Isao
中科院分区：
文献类型：
--
作者：
Fukuzawa, Seketsu;Takahashi, Saori;Suetake, Isao
5-甲基胞嘧啶(5mC)的氧化是由十-十一易位(Tet)酶催化生成5-羟甲基胞嘧啶(5HmC)及随后的氧化产物。被氧化的核苷酸是DNA去甲基化的中间产物，因为核苷酸是由胸腺嘧啶DNA糖基酶启动的碱基切除修复系统去除的。为了了解脱甲基化机理，需要一种简单、准确的方法在单碱基分辨下测定基因组DNA中的起始氧化产物5HmC。最近，我们发展了一种新的催化氧化反应，利用胶束包埋的氧化剂氧化5HmC生成5-甲酰胞嘧啶(5fC)，随后的亚硫酸氢盐测序可以确定5hmC在DNA中的位置。在本研究中，我们描述了催化氧化亚硫酸氢盐测序(COBS-SEQ)的优化及其在单碱基分辨率下定量分析基因组DNA中5HmC的应用。由于氧化步骤对基因组DNA的损伤很小，该方法允许我们缩小要分析的样品的规模。(C)2016爱思唯尔有限公司。保留所有权利。
Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed. (C) 2016 Elsevier Ltd. All rights reserved.