|Genome Mapping Section
DOE Human Genome Program Contractor-Grantee Workshop
62. A Resource of Mapped BAC Clones for Identifying Cancer Chromosome Aberrations
Norma J. Nowak1, Jeffrey
Conroy1, Greg P. Caldwell1, Joseph Catanese1,
Barbara Trask2, John D. McPherson3, David R. Bentley4,
Grace Shen5, and Pieter J. de Jong1
We are generating a resource of mapped BAC clones from the arrayed human BAC library (RPCI-11) for use in fluorescent in situ hybridization (FISH) analysis of chromosomal rearrangements in human tumors. This work is performed under the auspices of the NCI Cancer Chromosome Aberrations Project (CCAP). Our goal is to establish mapped BAC clones by screening 6-fold redundancy of the BAC library with 4,000 markers mapped at high resolution through radiation hybrid (RH) panels. Markers are judiciously selected spaced less than 1 Mb with preference for markers mapped to both high and low-resolution RH panels. To increase the success rate of the marker to BAC correlation, we are employing overlapping oligonucleotide probes ("overgo") based on the EST and STS sequences for the markers. The overgos are designed with the same average melting characteristics and are labeled by replicating the 5'overhangs using P-32 nucleotide triphosphates. To increase the throughput of the screening process to high density BAC colony membranes, the probes are pooled in mixtures of 36 probes each. Informative probe mixtures are prepared through the use of a 3-dimensional (6x6x6) probe pooling strategy consisting of 216 distinct probes. All predicted probe-BAC pairs are being confirmed by PCR and the expected 4-6 overlapping BACs for each marker are subsequently validated by restriction digest fingerprinting. In our initial mapping effort, we recovered BAC clones for 586 genetic markers on chromosomes 1, 5, 6, 18, 19, 21, 22 and Xp. After completion of three rounds of screening (736 markers), we have attained an average 2.5 Mb level of resolution and 4.2 BACs per marker. Overgos for the remaining Sanger framework markers are being designed and all mapped clones along with corresponding mapping information will be deposited in the public domain. Up to one BAC clone per marker will be characterized by in situ hybridization experiments to establish its usefulness as a FISH probe and to provide additional independent confirmation of the map location.
Work supported by NCI, DOE and the Wellcome Trust.
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