CONSTRUCTION AND EXTENSIVE CHARACTERIZATION OF A GOAT BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY WITH THREEFOLD GENOME COVERAGE

A goat Bacterial Artificial Chromosome (BAG) library of 61,440 indepen dent clones was constructed and characterized. The average size of the inserts was estimated at 153 kilobases by analyzing almost 500 clones using Not1 digestion followed by FIGE (Field Inverted Gel Electrophor esis) analysis. The library represents about three genome equivalents, which yields a theoretical probability of 0.95 of isolating a particu lar DNA sequence. After individual growth, the clones were arrayed in 30 superpools, which were organized in three dimension pools. A rapid technique for pool DNA preparation by microwave treatment was set up. This technique was compatible with PCR analysis. Primer pairs from 166 sequences (microsatellites, coding sequences from goat, and conserved Expressed Sequence Tags (ESTs) from humans) enabled the library to be successfully searched in 165 cases, with an average of 3.52 positive superpools. Only one sequence could not be found. The degree of chimer ism was evaluated by FISH analysis with DNA from over 110 clones and w as estimated at 4%. This BAC library will constitute an invaluable too l for positional cloning in ruminants, as well as for more general com parative mapping studies in mammals.