Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

The formation of 5-methyl-2′-deoxycytidine (5-MedC) following methylation of the C-5 position of cytosine in genomic DNA provides an epigenetic mechanism for the regulation of gene expression and cellular differentiation. We describe the development of a method using HPLC-ultraviolet (UV) detection for the accurate determination of 5-MedC in DNA. Genomic DNA was obtained from HeLa cells and rat liver tissue using an optimised anion-exchange column DNA extraction procedure incorporating a ribonuclease incubation step to remove any potential interference from RNA. Following extraction the DNA samples were enzymatically hydrolysed to 2′-deoxynucleosides using a combination of an endo-exonuclease plus 5′-exonuclease together with a 3′-nucleotidase. The hydrolysed DNA samples (10 μg on column) were analysed using narrow-bore reverse phase HPLC-UV detection. The level of 5-MedC in the DNA samples was expressed as a percentage of the level of 2′-deoxycytidine (dC) determined from calibration lines constructed using authentic standards for 5-MedC and dC. The percentage 5-MedC level determined for commercially available calf thymus DNA was 6.26%, for HeLa cell DNA was 3.02% and for rat liver DNA was 3.55%.