Date of Award

2000

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Supervisor

Christopoulos, T. K.

Rights

info:eu-repo/semantics/openAccess

Abstract

The ability of labeled DNA or RNA probes to bind with high affinity and specificity to complementary nucleic acid sequences forms the basis of nucleic acid hybridization assays. Immunoassays, on the other hand, exploit the strong and specific interaction between antigens and antibodies. One aspect of this dissertation deals with enhancement of the sensitivity of bioluminescence hybridization assays based on the photoprotein aequorin. This is achieved by introducing, enzymatically, multiple aequorin labels per DNA hybrid. The bound aequorin is determined by its characteristic bioluminescence. An 8-fold improvement in sensitivity is observed with the amplified assay as compared to an assay without the amplification step. Another aspect of this dissertation involves the development of two novel microtiter well-based DNA hybridization assays in which an expressible cDNA fragment encoding firefly luciferase serves as a reporter molecule. The reporter molecule undergoes in vitro expression through coupled transcription/translation or through sequential transcription/translation reactions. The bioluminescence activity of generated firefly luciferase was measured. The expression yield was improved with the separate transcription/translation protocol. In vitro expression provides signal amplification in hybridization assays because each cDNA label generates several enzyme molecules in solution. A third aspect of this dissertation combines the firefly luciferase cDNA and a cDNA encoding Renilla luciferase as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of synthesized firefly and Renilla luciferases were co-determined in a single sample based on the requirements of their characteristic bioluminescent reactions. In another aspect of this dissertation, the concept of in vitro expression as an analytical tool was further expanded in a novel microtiter well immunoassay which uses T7 RNA polymerase itself as the label. The enzyme label was determined by using the bound T7 RNA polymerase for in vitro expression of a firefly luciferase-coding DNA fragment. The activity of synthesized luciferase was measured. This is the first time that T7 RNA polymerase serves as the reporter molecule in hybridization assays or immunoassays.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2000 .L35. Source: Dissertation Abstracts International, Volume: 61-09, Section: B, page: 4700. Adviser: Theodore K. Christopoulos. Thesis (Ph.D.)--University of Windsor (Canada), 2000.

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