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Summary A method for the quantitative analysis of 5-methyl-2'-deoxycytidine (5mdC) in DNA based on enzymatic hydrolysis to nucleosides, separation and quantitation by GC/MS with internal standard has been developed. The method was applied to DNA from calf thymus and drosophila; the DNA from the Phage FX174 has been used to establish the accuracy of the procedure. The detection limit is 17 fmol 5mdC absolute and the content of modified cytosine in the DNA ofDrosophila melanogaster was shown to below that limit. Consequently, the genome contains less than one methylated cytidine per 330000 unmodified nucleosides or not more than 240 5mdC's overall, which renders an important function in regulation rather unlikely.

A method for the quantitative analysis of 5-methyl-deoxycytidine (5dmC) in DNA based on enzymatic hydrolysis to nucleosides, separation by Micro-HPLC and quantification with external calibration has been developed. The method was applied to DNA from calf thymus, drosophila and yeast; the fully sequenced DNA from the Phage FX174 has been used to establish the accuracy of the procedure. The detection limit is 0.4 pmol 5dmC absolute; the content of modified nucleoside in DNA of drosophila and yeast was shown to be lower than the current detection limit, which means that less than one methylated cytosine per 14000 (12000 resp.) unmodified nucleobases are to be found in the genomes.