A 5x genome coverage bovine BAC library: production, characterization, anddistribution

A bovine large-insert DNA library has been constructed in a Bacterial Artif icial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by trea tment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6 . Ln total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequ ency of clones without inserts is 4%. The chromosomal location of 51 BACs w as studied by FISH; 3 showed more than one signal, indicating a chimerism f requency of roughly 6%. Approximately 50% of the clones in the library cont ain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestio n of DNA prepared from 20 clones after serial culture for one and three nig hts. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high -density membranes, and PCR superpools were produced from the same set of c lones. Both membranes and superpools are available from the RZPD, Berlin (h ttp://www.rzpd.de). PCR 4-D superpools have been prepared from an additiona l 23,000 clones. The library has been screened for a total of 24 single-cop y sequences; positive clones have been obtained in all cases.