通过使用Peroxotungstate进行单无硫酸盐化学转换对5-甲基的基底分辨率分析
Gosuke Hayashi1, Kenta Koyama1, Hidefumi Shiota1
1Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Journal of the American Chemical Society
|November 3, 2016
概括
研究人员开发了一种新方法来检测DNA脱甲基化中的重要中间体5-Hydroxymethylcytosine. 这种技术使用氧化和测序来识别hmC的位置,提供了比现有方法更简单的替代方法.
科学领域:
- 表观遗传学和基因组学
- 分子生物学
- 生物化学
背景情况:
- 作为活性DNA脱甲基化的中间体,5-Hydroxymethylcytosine (hmC) 起着至关重要的作用.
- 精确检测hmC对于理解表观基因调节至关重要.
- 现有的hmC检测方法可能很复杂或缺乏基础分辨率.
研究的目的:
- 开发一种用于检测5-Hydroxymethylcytosine (hmC) 的基础分辨率新方法.
- 使用过氧化剂介导的氧化与hmC鉴定的测序分析.
- 为表观基因组分析提供一种简单的,不含二硫酸盐的技术.
主要方法:
- 开发了一种结合过氧化和DNA测序的方法.
- 在DNA聚合酶延长过程中研究了氧化hmC (三基胺,thT) 的行为.
- 采用桑格测序和Illumina测序来检测和映射hMC.
- 使用热循环对双链DNA进行优化过氧化状态处理.
主要成果:
- 氧化产物 hmC (thT) 被DNA聚合酶耐受,使其能够检测到.
- 桑格测序区分hmC与非甲基化细胞因子在单链DNA的氧化后.
- 热循环增强了双链DNA中的hmC氧化.
- 在合成基因组片段中,Illumina测序成功地确定了hmC的位置.
结论:
- 已经建立了一个新的,不含二硫酸盐的方法来检测5-Hydroxymethylcytosine (hmC).
- 这种技术简化了与hMC相关的表观基因信息的获取.
- 这种方法有望促进DNA脱甲基和表观遗传调节的研究.
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