YAC Library Screening I: Preparation of Hybridization Filters and PCR Pools

The storage of yeast artificial chromosome (YAC) libraries in ordered microtiter plates required a new approach to screening for clones containing specific DNA sequences. Screening libraries of some 60,000 clones by hybridization to filters prepared from individual 96-well microtiter plates was not a feasible option, prompting development of the polymerase chain reaction (PCR)-based screening approach of Green and Olson (1 ). Here (and in all subsequently developed PCR-based strategies), YAC libraries are screened by performing the PCR on a series of pools of DNA derived from specific mixtures of yeast clones. Amplification of target DNA sequence from an individual pool indicates the presence of the required YAC within the parent microtiter plates. Further rounds of testing on subsidiary pools are used to reveal the exact location of the YAC. Thus, a library of approx 36,000 clones may be prepared as 24 individual pools of 1536 YACs each for the first round of the PCR, each pool containing yeast DNA from 16 microtiter plates. Screening by the PCR therefore requires the preparation of pools of total yeast DNA derived from several thousand different YAC clones in equal amounts (2 –4 ). Pools containing fewer YACs may also be required for subsequent stages of PCR screening.