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 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%.
Sandhu
Jatinderpal
J
Cancer Biomarkers and Prevention Group, Biocentre, Department of Cancer Studies and Molecular Medicine, University of Leicester, University Road, Leicester LE17RH, UK.
Kaur
Balvinder
B
Armstrong
Christine
C
Talbot
Christopher J
CJ
Steward
William P
WP
Farmer
Peter B
PB
Singh
Rajinder
R
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000032:analysis; D016678:Genome; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D003841:Deoxycytidine; D004247:DNA; C016569:5-methyldeoxycytidine