Comparison of dot-blot and Northern blot hybridizations in the determination of genetic relatedness of United States bluetongue virus serotypes.

Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.