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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.
Progress with a strategy for cosmid closure of band 16q24.3
D.F. Callen, S. Apostolou, S. Lane, S.A. Whitmore, N.A. Doggett, R.I. Richards, J.C. Mulley, G.R. Sutherland
Centre for Medical Genetics Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006. Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
A strategy for contig closure, currently being implemented for band q24.3 of chromosome, involves the following steps: l. saturation of the region with cosmids identified by probing the high density chromosome 16 specific cosmid arrays with all cloned sequences known to map to this region. 2. Identification of contigs to which any of the cosmids identified belong. 3. Verification of contigs by STS development and mapping. 4. Contig extension by development of STSs from the ends of contigs and from the 3' and 5' ends of cDNAs and the re-screening of arrays with these. We have targeted 16q24.3 since it is a gene rich region of considerable biological interest since it possesses a major gene involved in breast cancer and other tumors. Alu-PCR products from two somatic cell hybrids in which the only human chromosome material was the distal portion of 16q, were used to probe high density grids of fingerprinted cosmids. Each cosmid isolated was mapped back to the region by Southern analysis of hybrid panel DNA. 49 cosmids, many of which are members of unmapped contigs, have now been located within 16q24. STSs from the ends of selected cosmids are being isolated by inverse PCR and being used to screen for additional cosmids, verifying contig structure and screening YACs. It is estimated that 1.8 Mb or 45% of this region is now in mapped cosmids and cosmid contigs. Direct selection has been used to isolate pools of cDNAs from cosmids. These cDNA pools are being used to screen the gridded cDNA filters of LLNL. We are aiming to produce a verified minimum tiling path of cosmids over the region as a set of reagents in preparation for large scale sequencing of this chromosome.
This work was funded by the DOE Genome Program Grant DE-FG02-89ER60863.