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Updated: Jun 22, 2026
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
Published on: October 5, 2012
Determination of 5-methyl-2'-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet
Jatinderpal Sandhu1, Balvinder Kaur, Christine Armstrong
1Cancer Biomarkers and Prevention Group, Biocentre, Department of Cancer Studies and Molecular Medicine, University of Leicester, University Road, Leicester LE17RH, UK.
Abstract:
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 microg 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%.
