On the Nature of the Deoxyribonucleic Acid-Methyl Green Reaction

Methyl green has frequently been used for histochemical and in vitro demonstration of deoxyribonucleic acid (DNA). I t has been reported that this color or staining reaction requires the presence of highly polymeric DNA (1) and that its molecular size influences the extent of the reaction. Thus, as the DNA is broken down by deoxyribonuclease (DNase), there is a corresponding decrease in the binding of the dye (1, 2). The reaction is greatly decreased when applied to DNA which has been broken down by ultraviolet (3) and by x-irradiation (4), by acidification (3), or by heating (1). I t has been deduced (5-8) that such treatments cause not only a diminution in length of the twin-helical chain (9) (i.e. degradation) but also a separation of the twin strands (denaturation). I t was therefore of interest to determine which of the structural or macromolecular features of the DNA are responsible for the staining reaction and whether degradation or denaturation (or both) lead to the diminished affinity for the dye. Such a study is feasible as it has been shown that the sonic treatment of DNA leads to extensive degradation unaccompanied by denaturat ion (5, 10-12). A solution (0.4 rag. per ml. of 0.2 ~t NaC1) of DNA, prepared from calf thymus by the method of Schwander and Signer (13), was subjected to sonic vibrations (9 kc. sonic oscillator, Raytheon Mfg. Co., model S102A) and aliquots withdrawn at different exposure times. This permitted the preparation of a graded series of DNA samples varying in weight average sedimentation coefficient from 21.3 to 5.5 S. This corresponds to a range of molecular weights from 6.7 X 106 to