Genomic subtractive hybridization to isolate species-specific DNA sequences in insects

Selective enrichment has been used in a number of instances for the isolation of species-specific sequences in prokaryotes. This paper reports the successful application of the technique to insects. Genomic probes were derived to the target species D. funebris and D. simulans. The method involves the biotinylation of non-target 'driver' DNA prepared from the closely related species D. melanogaster and its hybridization to homologous sequences in the target DNA. Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments. All DNA fragments isolated in the D. funebris experiments proved to be specific to that species. Five out of twenty-four fragments screened in the D. simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D. melanogaster.