Molecular hybridization of ribonucleic acid with a large excess of deoxyribonucleic acid

Abstract

When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA–RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA–RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2×SSC and 3×SSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2×SSC buffer. Rate-constants for DNA–RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA–RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90–95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.