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J Bacteriol. 1968 March; 95(3): 893-900
Copyright © 1967 American Society for Microbiology. All Rights Reserved.

Deoxyribonucleic Acid Homology in Bacterial Taxonomy: Effect of Incubation Temperature on Reaction Specificity

^a Department of Microbiology, University of Washington, Seattle, Washington 98105

ABSTRACT

Parameters affecting deoxyribonucleic acid duplex (DNA-DNA) formation on membrane filters were evaluated. The reference strains used were Cytophaga succinicans strain 8, which has a guanine plus cytosine (GC) content of 38%, and Myxococcus xanthus strain FB, which has a GC content of 70%. Both organisms are gliding bacteria classified among the myxobacteria. Among the parameters evaluated, the incubation temperature used during duplex formation was found to be the most important in terms of the physical nature of the reaction product. When an incubation temperature 25 C below the melting point (T_m) of the native DNA was used, homologous duplexes exhibited a thermal stability similar to that of native DNA. At 35 C below the T_m, a considerable proportion of the duplexes were of much lower stability; at 40 C below the T_m, most of the duplexes were of much lower stability. Similar duplexes of low stability were also formed between DNA molecules from morphologically and nutritionally diverse organisms, provided the GC percentages of the DNA preparations were similar. Competition between unlabeled and labeled DNA fragments for binding sites on immobilized DNA was also greatly influenced by the incubation temperature. Heterologous DNA-DNA complexes exhibited thermal stabilities which correlated with measurements of DNA homology in experiments involving competition. In addition, the difference in thermal stabilities of heterologous and homologous DNA complexes (T'_m) may provide a measure of divergence in nucleotide sequences.

¹ Present address: Department of Veterinary Science, Virginia Polytechnic Institute, Blacksburg, Va. 24060.

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