The formation of hybrid DNA molecules and their use in studies of DNA homologies

Abstract

Heavy-isotope-labeled DNA has been used to study strand separation and recombination. The rate at which 14 N− 15 N biologically half-labeled DNA can be made to separate into subunits corresponds very closely to what has been predicted for the rate of unwinding of the strands of a double helix. The use of a phosphodiesterase from E. coli ( Lehman, 1960 ) which selectively attacks single-stranded DNA has made it possible to remove unmatched single chain ends from renatured DNA and reduce the remaining differences between renatured and native DNA. A mixture of heavy-isotope-labeled and normal bacterial DNA was taken through a heating and annealing cycle, treated with the phosphodiesterase and examined by cesium chloride density-gradient centrifugation. Three bands were observed, corresponding to heavy renatured, hybrid, and light renatured DNA. As would be expected for random pairing of complementary strands, the amount of the hybrid was double that of either the heavy or the light component. It has thus been demonstrated that the strands which unite in renaturation are not the same strands that were united in the native DNA but instead are complementary strands originating in different bacterial cells. The formation of hybrids has been possible only where the heavy and normal DNA samples have a similar overall base composition. It has also been shown for DNA samples isolated from bacteria of different genera in a case where genetic exchange by conjugation has been demonstrated. The evidence for the parallelism between genetic compatibility and the formation of DNA hybrids in vitro has led to the proposal that organisms yielding DNA which forms hybrid molecules are genetically and taxonomically related.