Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR.

Yeast artificial chromosome libraries are extremely valuable for isolating large fragments of genomic DNA (1). One such library, which has been used extensively for screening purposes (2), has been made available to our laboratory and several others to maximize its use. This library, representing a fivefold coverage of the human genome, is currently being screened by a combination of PCR and hybridization protocols (3). Yeast colonies are grown on single filters in 24 x 16 gridded arrays. DNAs from master pools of five filters are screened, followed by screening of five single filter pools for each positive master pool. Finally, individual positives are identified by colony hybridization (3). This final step of colony hybridization is often difficult and time-consuming. We have devised a simple and rapid method for screening single filters by PCR of matrix pools, circumventing the need for colony hybridizations. Three copies of a single filter array known to contain a target YAC were grown as described earlier (3). The first filter was cut into 12 columns, each containing two lanes of colonies. The second filter was cut into 8 rows, each containing two lanes of colonies. Strips were cut with a scalpel blade and placed into 1.5 ml microfuge tubes using sterile forceps. To each tube was added 0.5 ml AHC culture medium [40 mM ammonium sulfate, 110 ,uM adenine hemisulfate, 0.17% (w/v) yeast nitrogen base without amino acids, 1 % casamino acids (w/v), pH 5.8]. Tubes were vortexed for several seconds and 10 Al was removed to a fresh tube; the remainder was frozen in glycerol for future screenings. To this was added 60 41 lysis solution [1