32P-base analysis of DNA

A ³²P-labeling method for the base composition analysis of nonradioactive DNA was developed consisting of the digestion of DNA to deoxynucleoside 3′-monophosphates by incubation with a mixture of micrococcal nuclease and spleen phosphodiesterase, transfer of ³²P-label from γ-³²P]ATP to the 5′-hydroxyl groups of the mononucleotides by T4 polynucleotide kinase, two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose of the resultant 5′-³²P]deoxynucleoside 3′,5′-bisphosphates, autoradiography, and scintillation counting. The method was standardized to afford quantitative digestion of DNA to mononucleotides as well as to give quantitative incorporation of ³²P-label into the nucleotides in the DNA hydrolysate so as to make the method an accurate means for determining the base composition of eucaryotic DNA containing adenine, guanine, thymine, cytosine, and 5-methylcytosine.