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

摘要

5-Hydroxymethylcytosine (~(hm)C) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring C by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of ~(hm)C, trihydroxylated thymine (~(th)T), 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 ~(hm)C 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 ~(hm)C in double-stranded DNA Furthermore, Illumina sequencing analysis of ~(hm)C-containing synthetic genome fragments enabled us to identify simultaneously the positions of C in base resolution. This bisulfite-free simple ~(hm)C detection technique could facilitate the acquisition of epigenomic information.