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

By J. Sandhu, B. Kaur, C. Armstrong, Christopher J. Talbot, William P. Steward, Peter B. Farmer and Rajinder Singh

Abstract

This is the Final Draft version of the article published: Sandhu, J., Kaur, B., Armstrong, C., Talbot, C. J., Steward, W. P., Farmer, P. B., Singh, R., 2009. Determination of 5-methyl-2 '-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, Vol.877, Issues 20-21, pp. 1957-1961.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%

Publisher: Elsevier
Year: 2009
OAI identifier: oai:lra.le.ac.uk:2381/7707

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