The present thesis work contributes to two areas of molecular genetics, namely the development of methods for identifying single nucleotides and the understanding of the mechanism by which antisense oligonucleotides inhibit eukaryotic gene expression. Three single nucleotide detection methods are presented. The first method utilizes hybridization with synthetic oligonucleotide probes to detect single or multiple nucleotide differences between alleles in genomic DNA without electrophoretic separation. Total genomic DNA (either digested or undigested with restriction endonucleases) was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. We show that this method can be used to discriminate the DNA of a HLA-B27-positive individual from the DNA of a HLA-B27-negative individual using a single HLA-B27 sequence-specific oligonucleotide probe. In situ dot analysis was also used to detect the presence of the sickle cell allele in DNA samples from normal donors (ßA/ßA), homozygous (ßS/ßS) and heterozygous (ßS/ßA) sickle cell donors using a pair of allele-specific oligonucleotide probes for the human ß-globin gene.
Two other methods are also capable of discriminating single nucleotide variations. They are based upon the action of DNA modifying enzymes (DNA ligases and DNA polymerases) on allele-specific oligonucleotide substrates annealed to the target DNA sequences. Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligonucleotides when guided by a complementary DNA template. When a single base-pair mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligonucleotides decreases. The highly specific nature of this ligation reaction provides the enzymatic basis for detection of single base-pair variations in a ligation amplification reaction (LAR). The product of successful ligation, guided by a completely complementary DNA template, is then amplified either linearly or exponentially using sequential rounds of template-dependent ligation. Similarly, the allele-specific polymerase chain reaction (ASPCR) takes advantage of the decreased rate of primer extension by DNA polymerase from Thermophilus aquaticus when a single mismatch exist at the 3' end of oligonucleotide primer after it is annealed to a DNA template. Two allele-specific primers differing by a single nucleotide and a third primer, downstream on the opposite strand can be devised such that each allele-specific primer set (consisting of an allele-specific primer and the down stream primer) will direct the amplification of a polymerase chain reaction fragment only in the presence of the allele-specific genomic DNA template. The present work demonstrates that both LAR and ASPCR can be used to distinguish between the sickle cell (ßS) and the normal ß-globin allele (ßA)
The second portion of the present thesis addresses the mechanism of antisense oligonucleotide inhibition of eukaryotic gene expression. We designed an eukaryotic expression system by introducing a human growth hormone (hGH) gene into a mouse L-cell line. We tested the ability of antisense oligodeoxyribonucleotides and oligomethylphosphonates, which are complementary to the splice junction, translation start site, and a stretch of poiypurine-polypyrimidine sequences suitable for triple helix formation, to inhibit the expression of the hGH gene. We show that the antisense oligonucleotide may operate by a mechanism other than the simple blocking of complementary DMA sequence in the gene of interest. Furthermore, we also demonstrate various confounding effects of oligonucleotide cytotoxicity on the overall antisense inhibition of hGH gene expression.
Antony J. Zuccarelli
R. Bruce Wallace
Barry L. Taylor
R. Bruce Wilcox
Donna D. Strong
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Nucleotides -- analysis; Oligonucleotides -- analysis; Eukaryotic Cells -- metabolism; Genetics, Biochemical
viii; 172 p.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Wu, Daniel Y., "The Use of Synthetic Oligodeoxyribonucleotides in Direct Analysis of Single Nucleotide Variations and Inhibition of Eukaryotic Gene Expression" (1990). Loma Linda University Electronic Theses, Dissertations & Projects. 1690.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives