Base ratio determination using unpurified DNA

Abstract

A technique is described for extracting DNA and measuring the mole per cent guanine plus cytosine of DNA from animals, bacteria, and plants. Cells were disrupted by lysis or homogenization, and in the case of animals and plants, total nucleic acids were then extracted. The extract, or in the case of bacteria the total lysate, was subjected to the combined effects of ribonuclease and alkaline hydrolysis to remove RNA. DNA was acid precipitated, collected, washed, and hydrolyzed in 0.1 M HCl at 100°. This quantitatively liberated guanine and adenine from the impure DNA, and the bases were separated by ion-exchange chromatography on Dowex 50. The mole fractions of guanine and adenine (and therefore mole per cent guanine plus cytosine) were then calculated from simple spectrophotometric determinations on the eluted peaks. The technique is of superior precision to those in present use, does not require purified and highly polymerized DNA, and should be capable of automation with commercially available liquid chromatographs.