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Genome Sequencing
Section
DOE Human Genome Program
Contractor-Grantee Workshop VII |
4. Physical Mapping and Sequencing of Human Chromosome 16p12.1-11.2 Hyung Lyun Kang,
Yicheng Cao, So Hee Dho1, Diana Bocskai, Mei
Wang, Xuequn Xu, Jun-Ryul Huh1, Byeong-Jae
Lee1, Francis Kalush2, Judith G.
Tesmer3, Eunpyo Moon4, Norman A.
Doggett3, Mark D. Adams2, Melvin I.
Simon, and Ung-Jin Kim The first goal of the Human Genome Project is to determine the nucleotide sequences of the entire human genome. We have been mapping and sequencing the 6 Mbp region near the 16pCEN on the short arm of human chromosome 16 (16p12.1-11.2) jointly with The Institute for Genomic Research (TIGR) and Los Alamos National Laboratory (LANL). As shown by the complete sequences from the BACs derived from this region, the target region has many small and large peri-centromeric repeats. It has been theorized that due to these repeats, many of which consist of large numbers of short tandem repeats, near-centromeric regions are difficult to clone and map. In fact, most genomic libraries tend to have fewer clones covering the centromeric and telomeric regions. Our target region is relatively sparsely covered by STS markers. In fact, most genomic libraries tend to have fewer clones covering the centromeric and telomeric regions. Our target region is relatively sparsely covered by STS markers. To provide large, contiguous stretches of BACs from the target region for high throughput shotgun sequencing at TIGR and JGI, Caltech has been developing BAC contigs using the STS and other ordered markers obtained from the YAC-STS map that was previously constructed by LANL. The 12X coverage human BAC libraries constructed at Caltech (A, B, and C) were screened by the combination of the STS-PCR screening on pooled libraries and the hybridization-based screening using probes that include cDNA inserts, BAC end clones, genomic DNA fragments and BAC inserts. Initially, a total of 46 STSs were screened against the libraries. More recently, Caltech has constructed a 7X coverage library D from approved human DNA samples, which has been screened by hybridization using the probes derived from STS-PCR products, BAC clone inserts (for BAC-to-BAC hybridization), and gel-purified YAC DNA (YAC-to-BAC hybridization). Thus far over 1,000 putative BACs from the target region have been identified. The clones are being built into overlapping contigs based on the analyses that include STS contents, BAC-to-BAC hybridization data, insert size, restriction fingerprint analysis, BAC end sequencing and BAC end sequence match with completely sequenced BACs, and FISH mapping on some selected BACs. Over 30 BACs from this region corresponding to approximately 4 Mbp in length have been sequenced at TIGR. To close the remaining gaps, we are currently designing new STS markers and OVERGO probes based on the BAC end sequence data along with the Alu-PCR products from the YAC clones covering the gaps. We also plan on screening new 4X coverage EcoRI BAC library. For the description, protocols, and data related to our projects, please visit our WEB site http://www.tree.caltech.edu. |
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