Screening a BAC Library With Nonradioactive Overlapping Oligonucleotide (Overgo) Probes

Comparative mapping has been the primary approach for analyzing genomes of divergent species because gene linkage is often conserved over evolutionary time, a phenomenon known as synteny. Comparisons are based on computational detection of orthologous sequences between species. Small overlapping oligonucleotide (overgo) probes, consisting of two self-annealing oligonucleotides at the 3′-end, are filled in with labeled deoxynucleotides. Overgo probes are designed against highly conserved regions between two genomes, at regular intervals (30–40 kbp). All methodologies reported to date have used radioactive dATP and dCTP to label overgo probes. We report a nonradioactive labeling protocol to produce digoxigenin-labeled overgo probes for high-throughput screening of the Actinidia chinensis bacterial artificial chromosome library.