The effects of mercury-substitution on the hybridisation characteristics of nucleic acids

Abstract

The effect of different levels of mercury substitution on the rate and extent of hybridisation of globin mRNA with a complementary DNA (cDNA) copy has been investigated. It was found that mercuration significantly reduces both the rate of hybridisation and the extent of the reaction, but that these effects are abolished when at least stoichiometric amounts of 2-mercaptoethanol are included in the hybridisation medium. As a preliminary to using this technique to isolate specific groups of sequences after long-term hybridisations, we have investigated both the rate of demercuration of RNA and its retention on thiol-sepharose columns after extended incubation under commonly employed hybridisation conditions at 43 degrees or 60 degrees. Retention was essentially quantitative even after incubation times of 300 hours at 43 degrees, but decreased significantly after 48 hours at 60 degrees. It is concluded that thiol-sepharose chromatography offers considerable advantages over hydroxyapatite chromatography for the recovery of hybridised sequences, particularly with regard to the lower levels of non-specific binding obtained and its ability to distinguish directly between DNA-DNA and DNA-Hg RNA hybrids.