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Sponsored
by the U.S. Department of
Energy Human Genome Program
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DOE Human Genome Program Contractor-Grantee Workshop IV
Santa Fe, New Mexico, November 13-17, 1994
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Introduction to the Workshop
The electronic form of this document may be cited in the following style: Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected. |
Construction of a High Resolution P1 and cDNA Map in the Down Syndrome Region of Chromosome 21Jan-Fang Cheng and Yiwen Zhu A high resolution clone map is constructed in the "major Down syndrome region" of chromosome 21 for a number of reasons: (l) transgenic studies of Down syndrome phenotypes (see abstract by Smith et al.), (2) identification of the mouse Weaver gene by complementation analysis. (3) DNA templates for production sequencing of the genomic fragments yielded interesting phenotypes. We have developed a two-tier hybridization method to screen a human P1 library (Du Pont Merck and Genome System Inc.) for clones in the region of 21q22.2-22.3. The goal is to construct a high resolution clone map providing continuous coverage of human DNA with P1 clones. A rapid initial screening of the P1 library is carried out by filter hybridization using pooled DNA probes derived from a specific chromosomal region approximately 3 Mb in length. We have included inter-Alu fragments derived from YACs, STSs, and mapped cDNA sequences in the probe pool to increase representation of the target region. The P1 filter contains DNA prepared from the pooled library (series B library) which was constructed by pooling 12 clones of the same row from the original library. The first tier hybridization therefore indicates the plate and row yielding positive clones. The twelve clones from each positive hybridization are re-gridded on filters for the second tier of hybridization using the same probe pool. The two tier screening produces a small set of clones (approximately 130 clones in a 3 Mb region with this 3.5 hit library) for contig construction. This process bypasses the slow and costly process of screening of the entire genomic library with every available STS. We have used this hybridization method to isolate 130 P1 clones in the major Down's syndrome (DS) region of chromosome 21 which comprises approximately 3 Mb of genomic DNA extending from D21S17 to ETS2. Overlaps between these P1's are determined using end probes generated from each P1 to cross-hybridize with DNA isolated from all isolates. This hybridization identifies overlapping P1's as well as gaps between two P1 contigs. The end probe which identifies a gap is used to probe the pooled P1 library again or to probe a chromosome 21 specific cosmid library for clones that could bridge the gap. These P1 were used to isolate novel cDNAs by hybrid selections, and some of the P1s were used in creating transgenic animals for phenotypic studies. These results should impact on future targets of the production sequencing program.
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