A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid

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Abstract
The color reaction of Dische (1930) between diphenylamine and deoxyribonucleic acid (DNA) was studied and modified. The principal modifications are to add acetaldehyde and to perform the reaction for several hours at 30[degree] instead of for 3-10 minutes at 100[degree]. The modified reaction is more sensitive and specific and less susceptible to interference by other compounds than is the reaction at 100[degree]. The color reactions of DNA preparations from calf thymus, Escherlchia coll and bacteriophage Ta were compared with that of 2-deoxyribose. For the 3 sources the ratios of moles of apparent deoxyribose to gram atoms of DNA phosphorus were almost identical and between 0.46 and 0.485. In the early stages of the diphenylamine reaction there is a liberation of inorganic orthophosphate from DNA. The amount of P thus liberated from calf-thymus DNA and from E coli DNA is approximately 25% of the total phosphate bridges between adjacent purine nucleotides in the polynucleo-tide chain. This value is consistent with the view that the purine and pyrimidine nucleotides are randomly distributed along the chain. Conditions for the quantitative extraction of DNA from bacteria were studied. Evidence on the mechanism of the DNA-diphenylamine color reaction is discussed and taken to indicate that neither free deoxyribose nor free [omega] -hydroxylaevulaldehyde are intermediates.