LARGE-SCALE CLONING OF HUMAN-CHROMOSOME 2-SPECIFIC YEAST ARTIFICIAL CHROMOSOMES (YACS) USING AN INTERSPERSED REPETITIVE SEQUENCES (IRS)-PCRAPPROACH

We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomi c DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostl y derived from the CEPH Mark I library. Alu-element-mediated PCR was p erformed for each pool, and amplification products were spotted on hyb ridization membranes (IRS filters). IRS probes for the screening of th e IRS filters were obtained by Alu-element-mediated PCR. Of 708 distin ct probes obtained from chromosome a-specific somatic cell hybrids, 85 % were successfully used for library screening. Similarly, 80% of 80 Y AC walking probes were successfully used for library screening. Each p robe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preli minary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric h alf of chromosome 2q was considered to be good since 31 of 34 microsat ellites and 22 of 23 expressed sequence tags that were chosen from chr omosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrar y generated by our experiments. We have identified a minimum of 1610 d istinct chromosome a-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome. (C) 1995 Academic Press, Inc.