Genomic 5-methylcytosine determination by 32P-postlabeling analysis

Abstract

A simple and sensitive method for the quantitation of 5-methyldeoxycytidine in DNA has been developed by the adaptation of the Randerath 32P-postlabeling technique. Nucleic acids were digested to 3′-monophosphate nucleotides, which were converted to 32P-labeled 3′,5′-bisphosphate nucleotides, the 3′-phosphate was cleaved by the action of nuclease P 1, and the resultant 5′-[ 32P]-monophosphate nucleotides were separated by two-dimensional thin-layer chromatography. Less than 1 μg of DNA was required for the precise quantitation of 5-methyldeoxycytidine content to a detectable limit of 0.01% of the total cytidine residues methylated. The genomic 5-methyldeoxycytidine content may thus be quantitated in tissue samples, small or selective cell populations, senescing or terminally differentiating cells, or DNA from any source. We report here, for the first time, the genomic 5-methyldeoxycytidine content of normal human bronchial epithelial and normal human pulmonary mesothelial cells. The chromatographic separation of all of the normal and some of the rare monophosphate deoxyribonucleotides and ribonucleotides has been characterized. Thus, 5-bromodeoxyuridine and the RNA contamination of DNA or the DNA contamination of RNA can also be quantitated during the same analysis.