The ultimate goal of this project is to sequence human chromosome 10. At the end of the current grant period, a rudimentary physical map will be generated as a result of the current """"""""STS content"""""""" mapping effort. This map will consist of 1400 YAC clones which are anchored by 300 STSs and 700 hybridization probes. Mathematical analyses predict that this set of YACs will cover 99.9% of the chromosome and will be ordered in 20 contigs with an expected size of 7.7 Mb per contig. This map will also have been integrated with the genetic and cytogenetic maps. For the renewal grant period, there are three specific aims. First, the STS content map will be validated. The collection of 1400 YACs, whose addresses have been identified by screening a 2-tier pool, will be colony purified, and re-screened with the collection of 300 STSs and 700 hybridization probes. This step will significantly improve the ordering of the 1,000 anchors. The physical mapping product at this stage will contain two parts: (1) an STS map at a density of one anchor per 150 Kb; and (2) a set of 1400 aligned YACs, which will provide an average resolution of 53 kb. The YACs will be arrayed according to their map positions and distributed to the research community. Any new chromosome 10 STSs will be conveniently mapped to this grid using a pooling strategy. Second, a high-resolution, sequence-ready map will be developed with bacterial artificial chromosomes (BACs) as the model. Initially, high- density BAC grids will be probed with inter-Alu products from a VAC contig covering a 5 Mb region from 10p11.2. The 200 identified BACs will be aligned using a """"""""clone-limited"""""""", non-random anchoring strategy, also known as """"""""sample-without-placement"""""""". STSs and hybridization anchors will also be placed on the BAC contigs in order to correlate the BAC contigs to landmarks and compare the STS content of the BAC and YAC contigs. After the test case is successfully completed. the effort will be scaled up for the entire chromosome. The maximum average resolution of the aligned BACs will be 13 kb. The ordered BACs will also be distributed to the research community. Third, one-pass sequencing of the ends of the 6,000 chromosome 10 BACs will be initiated. Multiplex sequencing techniques (Church and Kieffer- Higgens, 1988). which are being used for a large scale sequencing project at CRI, will be used for this purpose. Additional STSs can be developed from the sequence information if necessary. The sequences will also be searched against databases to identify CpG islands, control elements, exons, open reading frames and functional motifs. These mapped """"""""sequence tags"""""""", at a density of one per 13 kb, will provide a framework when the sequencing of the entire chromosome is initiated. During this renewal period, approximately 1.6 Mb of raw sequences will be generated for 4,000 """"""""tags"""""""".
| Ma, N S; Zheng, C; Benchekroun, Y et al. (1996) Characterization of a flow-sorted human chromosome 10 cosmid library by FISH. Cytogenet Cell Genet 74:266-71 |
| Moir, D T; Dorman, T E; Day, J C et al. (1994) Toward a physical map of human chromosome 10: isolation of 183 YACs representing 80 loci and regional assignment of 94 YACs by fluorescence in situ hybridization. Genomics 22:1-12 |
| Zheng, C J; Ma, N S; Dorman, T E et al. (1994) Development of 124 sequence-tagged sites and cytogenetic localization of 217 cosmids for human chromosome 10. Genomics 22:55-67 |
| Moir, D T; Dorman, T E; Xue, F et al. (1993) Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products. Gene 136:177-83 |
