Quantitation of 5-methylcytosine by one-dimensional high-performance thin-layer chromatography.
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.
Leonard
S A
SA
Department of Molecular Pharmacology and Therapeutics, School of Medicine, East Carolina University, Greenville, NC 27858.
Wong
S C
SC
Nyce
J W
JW
D044503:5-Methylcytosine; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D003412:Cricetulus; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry; D005092:Exonucleases; D006868:Hydrolysis; D008836:Micrococcal Nuclease; D009705:Nucleosides / Q000737:chemistry; D011116:Polynucleotide 5'-Hydroxyl-Kinase
D009705:Nucleosides; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D005092:Exonucleases; C022650:spleen exonuclease; D008836:Micrococcal Nuclease