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. / Medicine, Faculty of / Medical Genetics, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/6316 |
| Date | 11 1900 |
| Creators | Everson, Ted |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 12653097 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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