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A construct of overlapping yeast artificial chromosomes spanning a seven centimorgan region of human chromosome 8p22

University of British Columbia

An important goal of the Human Genome Project is the physical mapping of the human genome. This thesis describes the preparation of a set of overlapping yeast artificial chromosomes (YACs), spanning approximately a 7 centiMorgan (cM) region of chromosome 8p22, delimited distally by D8S550 and proximally by D8S552. This set of overlapping YACs, or YAC contig, is a useful contribution to the human genome physical map. It will have valuable applications for the identification of sequence ready clones and for the detection of disease genes that may be mapped to 8p22. To construct the YAC contig, the Whitehead Institute/ Massachussetts Institute of Technology (MIT) Center for Genome Research genomic database was searched for YACs containing markers that were previously localized to the region of interest. A singly-linked contig, WC8»1, was found, which identifies a set of 62 overlapping YACs within this region. Database information for these YACs was examined in order to exclude from analysis any YACs for which significant evidence of chimaerism was available. A subset of YACs was chosen for further analysis; these included eight YACs with no evidence for chimaerism, and two YACs with relatively weak evidence for chimaerism. DNA sequence from the insert ends (terminal sequence) of a number of these YACs was isolated by a modified bubble PCR protocol (Riley et al.,1990), a procedure that amplifies terminal sequences. These sequences were then used to develop new markers for the region. PCR was performed, using selected markers from WC8«1 and the new markers designed from terminal sequences. PCR amplification of markers in the set of ten YACs resulted in the identification of overlapping YACs, forming a contig that completely spanned the region of interest. In addition, terminal sequences from YAC 729el2 were found to be highly similar to the murine guanine nucleotide release factor 2 (GRF2) gene; a marker designed from this sequence was amplified in a human chromosome 5 somatic cell hybrid, localizing this putative human gene to chromosome 5.

Thesis/Dissertation

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2009-03-21

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http://hdl.handle.net/2429/6316

Medical Genetics

University of British Columbia

UBCV

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