Date of Award
Chemistry and Biochemistry
Christopoulos, T. K.
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Perhaps the most challenging current analytical problem associated with PCR is the determination of the starting quantity of target DNA. The objective of this research was to devise novel quantitative PCR assays for DNA and RNA which are automatable, highly sensitive and easily adaptable to the routine clinical chemistry laboratory. One project focused on the development of the first quantitative assay for prostate-specific antigen (PSA) mRNA based on co-amplification with a recombinant RNA internal standard (IS) and hybridization performed in microtiter wells. The PSA mRNA and the IS, differing only in a 24 bp centrally located sequence, contained the same primer recognition sites and generated amplification products of identical size. After reverse transcriptase-polymerase chain reaction, the products were captured through biotin/streptavidin, hybridized separately to specific digoxigenin-labeled probes and detected with alkaline phosphatase labeled anti-digoxigenin antibody and time-resolved fluorometry. Samples containing total RNA from prostate cancer cells amidst 1 mug of RNA from healthy cells were analyzed. The ratio of the fluorescence values obtained for PSA mRNA and IS was linearly related to the number of cells in the range of 4 to 3000 cells. The goal of the second project was to develop a dual-analyte chemiluminescence hybridization assay for simultaneous determination of both amplification products in the same well, thereby facilitating automation of quantitative PCR. Aequorin and alkaline phosphatase were used as reporter molecules. The proposed quantitative PCR assay could detect as low as 430 target DNA molecules and the linear range extended up to 315000 molecules.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1998 .V47. Source: Masters Abstracts International, Volume: 39-02, page: 0511. Adviser: T. K. Christopoulos. Thesis (M.Sc.)--University of Windsor (Canada), 1998.
Verhaegen, Monique Elise., "Novel approaches in quantitative polymerase chain reaction." (1998). Electronic Theses and Dissertations. 2877.