Investigators' Abstract) The long term objective of this research is to sequence human chromosome 10. The specific goal of this proposal is to construct a complete physical map of chromosome 10. Yeast artificial chromosomes containing large DNA fragments will be identified and then ordered following a high resolution genetic map, which will be constructed as a specific goal of this proposal. The ordered clones constituting this physical map will be the source of DNA for sequencing the chromosome. Specifically, a chromosome 10 YAC library will be constructed. This library, which will consist of 1,500 clones whose average insert size is 350 kb, will constitute the equivalent of three copies of chromosome 10. The probability of finding a particular chromosome 10 sequence in such a collection is 95%. When appropriate, subclones from YACs will be made and screened for polymorphisms which will be mapped on extended families so as to refine the pre-existing genetic map. Chromosome walking will be used to expand loci by an STS/PCR-based strategy. The end fragments of individual YACs will be amplified by inverse PCR, and STSs will be developed. To identify overlapping YACs, PCR of STSs will be performed on YAC pools and then subpools of those which test positive. Multiple, systematic screening for STSs in the YAC library will allow the contiguous sequences in different clones to be rapidly ordered. To help order YAC contigs, individual YACs will be assigned to chromosomal regions using a well-characterized panel of hybrid cell lines. YACs containing sequences which are homologous to mapped RFLP probes will be used as nucleation points for locus expansion since the relative positions of such probes are well defined. The orientation of the contigs on the chromosome will be deduced following the fine genetic map. Gaps between YAC contigs will first be filled by walking in cosmid libraries. The nature and extent of remaining gaps will be determined by long range restriction mapping. These gaps will then be filled by additional YAC cloning.
| 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 |
