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Quantitation of 5-methylcytosine by one-dimensional high-performance thin-layer chromatography
S A Leonard1, S C Wong, J W Nyce
1Department of Molecular Pharmacology and Therapeutics, School of Medicine, East Carolina University, Greenville, NC 27858.
Abstract:
A method for the quantitative analysis of DNA 5-methylcytosine by one-dimensional high-performance thin-layer chromatography using alkylamino modified silica (HPTLC-NH2) plates is described. The preparative method is simple, involving enzymatic digestion of DNA with micrococcal nuclease and phosphodiesterase II to 3'-monophosphate nucleosides, conversion by T4 polynucleotide kinase to 32P-labeled 3',5'-bisphosphate nucleosides, and chromatographic separation of nuclease P1-cleaved 5'-monophosphate nucleosides. The weak, basic anion exchanger property of the HPTLC-NH2 plate enables separation of multiple samples in one dimension, whereas traditional polyethyleneimine cellulose plates require development of individual samples in two dimensions for analysis of 5'-methylcytosine.