Liquid chromatographic determination of 5-methylcytosine in DNA with fluorescence detection

A novel method to determine the content of 5-methylcytosine in DNA precisely, sensitively and simply has been developed using liquid chromatography (LC) with fluorescence detection. DNA was mildly digested by cooperation of nuclease P1 and DNase I, then the generated 2′-deoxynucleoside 5′-monophosphates (dNmp) were specifically derivatized fluorometrically at the position of the phosphoric acid moiety with 5-dimethylaminonaphthalene-1-[N-(2-aminoethyl)]sulfonamide (dansylEDA), followed by the separation of fluorescent dansylEDA derivatives of dNmp on the reversed-phase partition column. The method was more than 10 times as sensitive as the conventional ultraviolet detection method.