A HUMAN GENOME YAC LIBRARY IN A SELECTABLE HIGH-COPY-NUMBER VECTOR

An experimental yeast artificial chromosome (YAC) library consisting of one genome equivalent of human DNA was prepared in a selectable high-copy-number (hcn) YAC vector. Screening for unique loci was accomplished by PCR of successively smaller DNA pools and by hybridization to high-density microcolony blots. Inserts averaged 200 kb in size, but several YACs with inserts averaging about 650 kb were obtained when polyamines were added prior to yeast transformation. YACs were identified for 17 out of 29 sequence-tagged sites (STS) screened by a PCR-based approach. All YACs in the size range of 100-600 kb that were examined could be obtained at significantly elevated copy numbers following growth of the clones in methotrexate/sulfanilamide/thymidine-supplemented medium. The hcn YACs could also be selected during growth in microtiter dishes, and the resulting clones were used to prepare high-density microcolony DNA blots for hybridization with radiolabeled PCR products. DNA pools for the PCR-based screening of this experimental library are available to investigators interested in applications of hcn YACs.