Physical maps provide rapid access to any chromosomal region of interest, and serve
as essential tools for the localization of genes. Of particular interest is the observation that gene clustering occurs in telomeric areas, although few genes have been mapped to the telomeric end of human chromosome 8p. In an effort to refine the physical map of 8pter-8p23, radiation hybrid mapping was used to determine the chromosomal order of ten sequence tagged sites (STSs) mapping to this interval. Four out of ten loci were positioned
at 1,000:1 odds against order inversion, corroborating previously established linkage data. Secondly, STS content mapping in yeast artificial chromosomes (YACs) aided in the
identification of potentially overlapping YACs, and a preliminary YAC contig of the region was generated. Six YACs constituting a "minimum tiling path" were selected.
As cosmids provide the necessary reagents for detailed positional cloning strategies
and genomic sequencing, the primary objective of this project was to isolate bins of cosmids mapping to 8pter-8p23. To achieve this goal, Alu PCR products derived from the minimum tiling path YACs were used as hybridization probes against a chromosome 8 specific cosmid library. The initial screening identified an average of over 300 cosmids per YAC. However, when a subset of positive cosmids was digested with EcoRl, Southern blotted and re-probed with the Alu PCR products, a false positive rate of 55% was observed. Probing the blots with total human DNA detected numerous fragments containing repetitive DNA. As a final analysis to confirm the identity of specific clones, cosmid EcoRl fragments were hybridized onto Southern blots containing EcoRl digested YAC DNA. Fragments which clearly demonstrated sequence homology to the corresponding YAC were strongly positive with the YAC Alu PCR probe but negative with the total human DNA probe in the preceding
experiment, suggesting that unique sequences were being isolated. In contrast, sequence
homology was not demonstrated by fragments that were strongly positive with both the Alu
PCR and total human DNA probes. A single cosmid was also identified which confirmed
the overlap relationship between two YACs that was originally suggested by STS content
mapping. / Medicine, Faculty of / Medical Genetics, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/5881 |
| Date | 05 1900 |
| Creators | Ma, Lingli |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 9809500 bytes, application/pdf |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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