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Physical mapping and clone isolation from the telomeric region of human chromosome 8p

University of British Columbia

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.

Thesis/Dissertation

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2009-03-11

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http://hdl.handle.net/2429/5881

Genetics

University of British Columbia

UBCV

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