Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics

Abstract

Differences in the RNA-driven hybridization kinetics of genomic DNA and cDNA probes led us to examine physical parameters affecting these reactions. Cloned cDNA complementary to serum albumin (SA) mRNA hybridized in accordance with single component kinetics, whereas cloned SA genomic DNA hybridized more slowly and with multiple component kinetics. This difference is largely attributable to the relatively short and variable lengths of the mRNA complementary regions in the cloned genomic DNA. The rate of mRNA driven hybridization is affected to about half the extent observed for DNA renaturation as Na+ is increased or decreased from 0.18M. In the annealing of nucleic acids of high sequence complexity, after approximately 70% of reaction has been reached, the rate of the reaction is slowed and completion is not reached under "static" conditions. In practical terms, this is not the case for systems of low sequence complexity. This problem can be largely overcome by continuous or frequent mixing of the reactants, so that complex cDNA probes are hybridized essentially to completion, and kinetics can therefore be more readily compared to simple complexity standards.