Chicken BAC library screening using conventional and OVERGO probes

To align the developing chicken BAC contig physical maps with the existing linkage map, it's necessary to identify BACs corresponding to the DNA-based markers on the latter. Chicken BAC libraries, derived from DNA of a single UCD001 inbred Red Jungle Fowl, have been generated by our collaborators. Characterization of the first BAC library based on BamHI partial digest fragments initially was done by filter hybridization with pools of labeled, PCR-amplified fragments based on marker or gene DNA sequences. Individual marker/BAC assignments were made by Southern hybridization of BAC DNA with individual marker probes and/or PCR analysis. In this manner, 31 markers from 9 linkage groups generated 71 BamHI BAC candidates (2.3 clones per locus). This approach is labor- and cost-intensive. Thus, we began using pools of overgo probes, that are complementary synthetic oligonucleotides extended in vitro to generate ~40 base pair, double-stranded DNA probes. Two BAC libraries were hybridized to pools of 36 overgos derived as 4-dimensional subsets from 216 total probes. Each probe is present in a unique combination of any three dimensions with the redundant fourth dimension serving to improve accuracy. In screening the BamHI BAC library, 170 probes, among the 216 from GGA1 and GGA2 loci, generated 479 BAC clone assignments (or 2.8 per locus). In screening the EcoRI BAC library, 216 probes, designed to cover 35 linkage groups, produced 690 BAC clone assignments (3.7 per locus), with 14% of the overgos generating no positives. Selected BAC assignments are now being examined via cytogenetic (FISH) mapping.