HUMAN BAC LIBRARY - CONSTRUCTION AND RAPID SCREENING

We have constructed a human genomic bacterial artificial chromosome (B AG) library using high molecular weight DNA from a pre-pro-B cell line , FLEB14-14, with a normal male diploid karyotype. This BAC library co nsists of 96 000 clones with an average DNA insert size of 110 kb, cov ering the human genome approximately 3 times. The library can be scree ned by three different methods. (1) Probe hybridization to 31 high-den sity replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two South ern blot filters to which 31 HindIII digests of the pooled 3072 BAC cl ones were loaded. This identifies a particular HDR filter for which fu rther probe hybridization is performed to identify a particular clone( s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applie d to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screeni ng is particularly powerful since it allows us to isolate a desired BA C clone(s) within a day or so. The theoretical consideration of the ad vantage of this method is presented. Furthermore, we have adapted Vect orette method to our BAC library for the isolation of terminal sequenc es of the BAC DNA insert to facilitate contig formation by BAC walking . (C) 1997 Elsevier Science B.V.