Directed cosmid isolation of bovine markers for physical assignment by fish

A directed PCR-based iterative screening protocol was developed to isolate cosmids containing microsatellite markers linked to chromosomal regions of interest, such as those near the ends of linkage groups or quantitative trait loci. This method was optimized for large-scale screening of total genomic libraries and used to purify bovine cosmids that anchor the ends of the bovine linkage group 28 (BTA28). Cosmids containing ms markers RBP3 and BMS2060 were purified for fluorescence in situ hybridization and assigned to 28q18-q19 and 28q12, respectively. These assignments indicated that approximately 73% of BTA28 (90% excluding the centromere) is covered by the current linkage map. Since this method is applicable to any target gene sequence suitable for PCR amplification, it may be extended to comparative mapping of genes.