Quantitative determination of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A method to separate the four major bases (cytosine, guanine, thymine and adenine) and the two minor modified bases (5-methylcytosine and 6N-methyladenine) in DNA has been developed. For optimal separation, several different buffer systems are available for isocratic elution. The 12 5-methylcytosine (5-mC) residues in the plasmid pBR322 can be determined with a deviation of less than 3% of the expected value and have been used for internal standardization. Formic acid hydrolysis of bases and probably of DNA does not lead to the deamination of cytosine or 5-mC and thus can be used routinely for DNA hydrolysis. Adenovirus or baculovirus DNA does not contain detectable amounts of 5-mC. The distribution of 5-mC in hamster cell DNA appears to be nonrandom in that different 5'-CpG-3'-containing restriction sites are methylated to different extents.