Date of Award

2002

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Biology, Molecular.

Supervisor

Taylor, K.,

Rights

info:eu-repo/semantics/openAccess

Abstract

One aspect of this dissertation involves the investigation of T7 RNA polymerase (T7RP) as a label for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high detectability and reproducibility. Another aspect of the dissertation deals with the design of universal heterobifunctional linkers between antibodies and reporter genes for immunoassay development. The linkers consist of avidin or streptavidin conjugated to the oligonucleotide (dA)40. (Strept)avidin interacts with a biotinylated detection antibody whereas the oligonucleotide hybridizes with a complementary poly(dT) tail added enzymically to the 3' end of the reporter gene. The linker is evaluated in a model two-site (sandwich-type) immunoassay performed in microtiter wells. A 4.3 kb plasmid containing the firefly luciferase cDNA is used as a reporter. Upon in vitro expression of the reporter gene, multiple enzyme molecules are generated and detected in solution. In another study, we investigated five strategies for immobilization of a DNA probe to polystyrene microtiter wells for hybridization assays. Probes were immobilized: (a) through digoxigenin-antidigoxigenin interaction, (b) through biotin-streptavidin interaction, (c) by preparation and immobilization of oligonucleotide-albumin conjugates, (d) by activation of the polystyrene surface with poly (Lys, Phe) and subsequent conjugation with the probe, (e) by direct coating via physical adsorption of the probe to the wells. The hybrids were detected by indirect labeling through biotin-streptavidin or digoxigenin-antidigoxigenin and using alkaline phosphatase as a reporter molecule. We also developed a dual-analyte microtiter well-based chemiluminometric assay for genotyping single point mutations. The IVS-1-110 mutation of the human beta-globin gene (responsible for beta-thalassaemia) was studied as a model. Genomic DNA was extracted from whole blood followed by PCR amplification. The oligonucleotide ligation assay was performed on the amplified DNA using a common probe and two allele-specific probes. Ligation products were analyzed by a microtiter well-based assay in which aequorin and alkaline phosphatase are used as labels. The assay format enables simultaneous detection of normal and mutated allele in a single microtiter well.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2002 .T37. Source: Dissertation Abstracts International, Volume: 64-01, Section: B, page: 0082. Advisers: K. Taylor; T. K. Christopoulos. Thesis (Ph.D.)--University of Windsor (Canada), 2002.

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