Determination of cytosine, 3-methylcytosine, and 5-methylcytosine in nucleic acids by high performance liquid chromatography.

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A sensitive and selective analytical method for cytosine, 3-methylcytosine, and 5-methylcytosine in nucleic acids by high performance liquid chromatography was developed. This method involves hydrolysis of deoxyribonubleic acid (DNA) or ribonucleic acid (RNA) with perchloric acid at 100°for 1 hr, removal of the acid by weakly basic anion exchange resin (CG-4B, OH-form), separation of cytosine, 3-methylcytosine, and 5-methylcytosine from other bases by treatment with Dowex 1×2 column (HCOO-form), and the analysis of the eluate by high performance liquid chromatography. Recoveries were more than ninety percent. This method was applied to several nucleic acids, and molar ratio of 3-, and 5-methylcytosine to cytosine was obtained.

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