Jan-Fang Cheng, Steve Lowry, Duncan Scott, Yiwen Zhu and Eddy Rubin
Human Genome Center, Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
The LBNL Human Genome Center has focused its production genomic sequencing on the distal long arm of human chromosome 5. This region was chosen because it contains a cluster of growth factor and receptor genes and is likely to yield new and functionally related genes through long range sequence analysis.
Our mapping goal is to generate sequence-ready templates completely covering the target region. Templates would include clones isolated from human P1, PAC and BAC libraries. There are three key steps in our mapping strategy. First is the use of inter-Alu fragments generated from chromosome 5 non-chimeric YACs to isolate regionally specific clones. Second is to establish overlaps and orientations of the isolated clones. Overlaps between P1s, PACs and BACs were detected in filter hybridization using probes generated by vector-Alu PCR. Contigs were further oriented using STSs developed from known genes, ordered markers, and ends of P1s, PACs, BACs and YACs. The third step is to close gaps and verify the integrity of the cloned fragments. DNA sequences flanking gaps were used to identify additional clones for gap closure. Comparison between restriction fragments of genomic and cloned DNA allows us to sample the integrity of the cloned fragments.
Eighty-four non-chimeric YACs spanning approximately 42 Mb of the distal long arm of chromosome S were identified using fluorescent in situ hybridization (FISH). They formed 10 contigs which range in size from less than 2 Mb to approximately 9 Mb. These YACs were the major source of DNA for generating probes to identify P1, PAC and BAC clones in this region.
Two hundred seventy-four P1s, 53 PACs and 42 BACs were mapped to a 10 Mb region of Sq31 which contains the interleukin gene cluster and its distal 9 Mb of DNA. Of these clones, 223 P1s and all of the PACs and BACs were sized by using pulsed-field gel (PFG) electrophoresis. Chromosomal location of these clones were further confirmed by using FISH. Over 146 STSs derived from the end sequences of P1s, PACs and BACs were used to determine orientation of the clones and orientation of the contigs. The end STSs were also used in gap closure. A subset of these mapped clones were used as templates for production sequencing.
We are in the process of expanding the high resolution clone map to cover all of Sq3. Another 157 P1s, 21 PACs and several hundred BACs were isolated using probes derived from YACs located on the distal portion of Sq3. These clones have been assigned to specific regions of YACs, sized by PFG, and their location on chromosome S was confirmed by FISH.
Supported by a grant from the DOE under contract DE-AC03-76SF00098.
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