DIRECTED COSMID ISOLATION OF BOVINE MARKERS FOR PHYSICAL ASSIGNMENT BY FISH

A directed PCR-based iterative screening protocol was developed to iso late cosmids containing microsatellite markers linked to chromosomal r egions of interest, such as those near the ends of linkage groups or q uantitative trait loci. This method was optimized for large-scale scre ening of total genomic libraries and used to purify bovine cosmids tha t anchor the ends of the bovine linkage group 28 (BTA28). Cosmids cont aining ms markers RBP3 and BMS2060 were purified for fluorescence in s itu hybridization and assigned to 28q18-q19 and 28q12, respectively. T hese assignments indicated that approximately 73% of BTA28 (90% exclud ing the centromere) is covered by the current linkage map. Since this method is applicable to any target gene sequence suitable for PCR ampl ification, it may be extended to comparative mapping of genes.