Sensitive and label-free discrimination of 5-hydroxymethylcytosine and 5-methylcytosine in DNA by ligation-mediated rolling circle amplification

We develop a label-free fluorescence method for sensitive discrimination of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) based on the specific oxidation of 5hmC to 5-formylcytosine (5fC) and the subsequent conversion to uracil (U) in combination with ligation-mediated rolling circle amplification (RCA). This method exhibits high sensitivity with a detection limit of as low as 34.8 fM. It can discriminate as low as the 0.01% 5hmC level from the mixture, and exhibits good performance in serum sample analysis as well.