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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
A Human DNA Library in Bacterial Artificial Chromosomes (BACs): Application to Physical Mapping of Chromosome 22
Hiroaki Shizuya, Ung-Jin Kim, Bruce Birren, Tania Slepak, Valeria Mancino, April Mengos and Melvin Simon
Division of Biology, 147-75, Caltech, Pasadena, California 91125
Two new BAC vectors have been constructed. They are derived from the original pBAC108L vector. pBeloBAC11 has a cloning site in the lacZ, therefore BACs are identified as white colonies. The positive selection pTrimBAC111 is based on the ability of growing in the presence of trimethoprim in the thyA mutant bacteria when DNA insertion occurs. Using these vectors, we have constructed total human BAC library containing 96,000 clones with average insert size of ~125kb (i.e., 4X coverage).
The library can be accessed by hybridization of clones gridded at high density onto nylon filters in 5 x 5 configuration or by PCR with STS primers from BAC DNA of pooled clones. The library has been successfully probed with many known markers, YACs, Fosmids, and cosmids. Moreover, regions of the genome that are difficult to clone and maintain in YAC or cosmid vectors have been identified on BACs. Thus it appears to cover extensive regions of human DNA. Using the library we are constructing a high resolution physical map of human chromosome 22. One approach toward this goal is to identify as many chromosome 22 specific BACs as possible by probing with a variety of chromosome 22 markers and to walk from these anchor BACs. BAC inserts can be used to walk by hybridization in order to generate a long contig nucleated by the inserts (BAC to BAC walking). Fingerprinting these clones by restriction enzymes then confirms the contig, and identifies BACs located at the end of contigs for the next round of walking. After two rounds of BAC-to-BAC walking we collected more than 1,000 chromosome 22 specific BACs and constructed 69 separate contigs. Each contig spans about 300 kb on average, and therefore 20 mb of chromosome 22q has been covered, leaving, on average, a 350 kb gap between contigs. We are currently filling the gaps with a variety of methods.