Studies on the Kinetics of Renaturation of DNA

Citation

Wetmur, James Gerard (1967) Studies on the Kinetics of Renaturation of DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/YB2H-T758. https://resolver.caltech.edu/CaltechETD:etd-09232002-113015

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. A temperature jump system is described for studying fast DNA renaturation reactions. The reaction is found to be second order as seen in the time course of the reaction and in the concentration dependence of the rate. The stepwise base-pairing model of Saunders and Ross is extended to allow varying DNA base composition. Rate constants calculated with this model are compared with experimental rate constants at varying temperatures. T4 and T7 DNAs were fragmented by various procedures. The molecular weight of denatured DNA was determined by alkaline sedimentation. For a given DNA, fragmented into different molecular weights, the rate of renaturation is found to be proportional to the square root of the molecular weight. The rate of renaturation of DNA was measured in sucrose, glycerol, ethylene glycol and sodium perchlorate solutions. The melting temperature of DNA is changed by different amounts in each solvent. Nevertheless, the rate constant multiplied by the viscosity and divided by the renaturation temperature is found to be a constant. Thus, the rate determining step must be hydrodynamically limited. The complexity of the DNA of an organism is defined as the total DNA complement of the organism. The rates of renaturation of SV40, T7, Nl, T4, E coli and Ascites tumor DNA (non-repeated sequences) are inversely proportional to the complexity. After complexity correction, the rate of renaturation is found to depend slightly on the GC content of the DNA. The stepwise renaturation model predicts this result. A method is described for positively staining electron microscope grids prepared by the method of Kleinschmidt and Zahn. Some properties of N1 DNA are described. The DNA has a buoyant density corresponding to 64% GC, a molecular weight of 33 X 10[^6]and the property of reversible cyclization like lambdoid phage DNAs.

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