Isolation and Characterization of DNA-DNA and DNA-RNA Hybrid Molecules Formed in Solution

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na₂HPO₄ DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na₂HPO₄, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (t_m) obtained for SV40 DNA were 76 °C at 1 × SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 °C at 0.1 × SSC, and for NIH/3T3 DNA 82 °C at 1 × SSC and 68 °C at 0.1× SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 °C. The melting temperature of this hybrid in the annealing solution was 87 °C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 °C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to SI nuclease.