Base-Resolution Analysis of 5‑Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate

• Published in: Journal of the American Chemical Society Vol. 138; no. 43; pp. 14178 - 14181
• Main Authors: Hayashi, Gosuke, Koyama, Kenta, Shiota, Hidefumi, Kamio, Asuka, Umeda, Takayoshi, Nagae, Genta, Aburatani, Hiroyuki, Okamoto, Akimitsu
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.11.2016
• Subjects: 5-Methylcytosine - analogs & derivatives; 5-Methylcytosine - chemistry; Base Pairing; Base Sequence; DNA - chemistry; DNA - genetics; Selenium Radioisotopes; Sulfites - chemistry; Tungsten Compounds - chemistry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.6b06428

5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of hmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of hmC in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.