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Alkali Denaturation of Covalently Closed Circular Duplex Deoxyribonucleic Acid

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      The alkaline pH range over which the double stranded circular DNA of the replicative form of the bacterial virus, ϕX174, is reversibly denatured has been determined in order to specify accurately the conditions for a preparative procedure for this DNA based on selective alkali denaturation. The denaturation remains entirely reversible to pH 12.6 at an ionic strength of about 0.25 over a range in which the sedimentation coefficient, after an initial drop, more than doubles. The sharp transition above pH 12.6 in which the denaturation becomes completely irreversible is accompanied by only a small further increase in sedimentation coefficient. Sedimentation-pH titration curves have been determined in detail for the replicative form of ϕX174 (molecular weight 3.4 x 106 daltons) and for the circular DNA of a penicillinase plasmid of Staphylococcus aureus (molecular weight 16.5 x 106 daltons). The latter curve is displaced to a lower pH and the two phase nature of the curve in the range of increasing sedimentation coefficient is markedly accentuated.

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