The detection of subtle differences between different orthopoxvirus genomes by heteroduplex analysis

Corresponding DNA fragments from variola (Harvey) and monkeypox (Denmark) viruses which had been cloned into different plasmid vectors were subjected to heteroduplex analysis. Characteristic deletion loops corresponding to differences between the cloning vectors served as internal markers to identify and to orientate the heteroduplexed molecules. Partial denaturation of the resulting heteroduplexes was used as a primary screen to locate regions of heterogeneity between the poxvirus inserts. The denaturation threshold for homoduplexes was consistently higher than that for heteroduplexes. However, significant sequence divergence between corresponding fragments was indicated by larger than usual differences in thresholds between corresponding homo- and heteroduplexes. Denaturation bubbles of 0.1–0.5 kb were detected and hence small regions of heterogeneity between the genomes (180 kb) of variola and monkeypox viruses were localised. This procedure has a general application in comparative studies on large, complex but closely related DNA molecules.