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

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.