Rapid determination of methylated purines in DNA treated with N-methyl-N-nitrosourea using high-performance liquid chromatography.

A method for determining methylated purine bases in [3H]N-methyl-N-nitrosourea (MeNOUr) treated DNA is described. The method combines reversed-phase high performance liquid chromatography (HPLC) of methylated DNA after hydrolysis in dilute acid with the determination of radioactivity in the fractionated eluates. The peaks of the respective methylated purines were indentified by the use of internal standards. The method allows quantitative separation of 3-methyl-adenine (m3Ade), 7-methyl-adenine (m7Ade), 3-methyl-guanine (m3Gua), 7-methyl-guanine (m7Gua) and O6-methyl-guanine (m6Gua) within 20 min. Thus the total time required for determination of methylated purines is limited only by radioactivity measurements in the respective fractions.