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

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Abstract
The effect of different levels of mercury substitution on the rate and extent of hybridisation of globin mRNA with a canplenentary DNA (cDNA) copy has been investigated. It was found that nercuration significantly reduces both the rate of hybridisation and the extent of the reaction, but that these effects are abolished when at least stoichiomtric 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 thicl-sepharose columns after extended incubation under commonly employed hybridisation conditions at 43° or 60°. Retention was essentially quantitative even after incubation times of 300 hours at 43°, but decreased significantly after 48 hours at 60°. 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.