DETERMINATION OF BASE COMPOSITION OF DEOXYRIBONUCLEIC ACID BY MEASUREMENT OF ADENINE/GUANINE RATIO

A method is described for determination of the base composition (as guanine-tcytosine or adenine-tthymine content) of DNA by accurate measurement of the adenine/guanine ratio. The DNA is hydrolyzed with 0.03 ri-hydrochloric acid for 40 min. to release the purines. The hydrolysate is subjected to ion-exchange chromatography on Zeo-Karb 225. Apurinic acids are eluted with 0.0N-hydrochloric acid and then guanine and adenine are eluted separately with 2 N-hydro-chloric acid. Guanine and adenine are each collected as a single fraction, and the amount of base in each case is determined by measuring the volume and the extinction at suitable wavelengths. For use in the calculations, millimolar extinction coefficients in 2 N-hydrochloric acid of 12.09 for adenine at 262 mu, and 10.77 for guanine at 248 mu, were determined with authentic samples of bases. The method gives extremely reproducible results from 12 determinations with calf thymus DNA the adenine/guanine molar ratio had a standard deviation of 0.011; this corresponds to a standard deviation in guanine-tcytosine content of 0.2% guanine-fcytosine.