Chemical oxidation-free site-specific 5-hydroxymethylcytosine assay

Abstract

5-hydroxymethylcytosine (5hmC) shows a very close correlation with various diseases. Accurate quantification of 5hmC is of great significance for revealing its function in different tissues. Here we proposed a chemical oxidation-free and site-specific 5hmC assay. First, T4 β-glucosyltransferase (T4 β-GT) and AbaSI restriction endonuclease were used to cleave the double-strand DNA at the 3' end side away from the 5hmC to produce the sticky end. Then, a hairpin probe was ligated to the sticky end of target 5hmC as an adapter. Finally, polymerase chain reaction (PCR) amplification was employed to amplify the ligation products to achieve a high sensitivity determination of target 5hmC. The selectivity of the resulting assay is up to 4000-fold and 540-fold to distinguish 5hmC from cytosine (C) and 5-methylcytosine (5mC), respectively. The detection limit of the proposed 5hmC assay is 0.89 fM. This method can detect not only the content of site-specific 5hmC but also the percentage of site-specific 5hmC.