A microanalytical procedure for determination of the base composition of DNA

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Abstract
A new procedure for the determination of the percentage guanine plus cytosine (% G + C; mol/100 mol) values of microquantities of DNA is described. Its principle is a DNA-polymerase-I-directed nick translation of DNA in the presence of dGTP, dTTP, [3H]dCTP, and [.alpha.-32P]dATP. Kinetics experiments indicate that the plateaus value is reached in .apprx. 20 min of incubation under these experimental conditions. Percentage G + C is obtained from the linear relation 1/(% G + C) = 0.01 K[32P]/[3H] + 0.01, where the ratio of trichloroacetic-acid-precipitable radioactivity is taken into account, the K value being determined for each experiment by using a few reference DNA species of know composition. This procedure has proven suitable for analysis of plasmidic, viral and cellular DNA species of different base composition (25-75% G + C), shape (linear and circular double-stranded DNA) and size (100-150,000 base pairs). Usual methods for % G + C analysis (buoyant density and melting temperature determinations) yield unreliable results in the presence of either modified or unusual bases: the double-labeling procedure is still valid under these conditions. The latter is, therefore, the method of choice for analysis of rare DNA species which are available in very small quantities (it requires amounts of DNA as low as 1 ng, i.e., several order of magnitude lower than those used for chromatographic analysis of DNA hydrolysates). Since the obtention of highly purified DNA is an essential prerequisite for the double-labeling procedure, a method for purification of bacterial DNA is detailed in the present work.