Date of Award
Chemistry and Biochemistry
Christopoulos, T. K.
CC BY-NC-ND 4.0
The molecular hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph$\sp1$) which arises from the reciprocal translocation of the c-ABL proto-oncogene and the BCR gene. Expression of the chimeric fusion gene results in 8.5 kb leukemia-specific BCR-ABL mRNA transcripts. The ability to detect the disease early on in its progression is key to survival. To facilitate the diagnosis of de novo leukemia and the monitoring of patients with CML, highly sensitive, nonisotopic assays were developed for the determination of CML-specific BCR-ABL mRNA in leukocytes. The methods involved reverse transcription of mRNA followed by PCR amplification of only the diagnostically significant BCR-ABL cDNA. The determination of PCR products was performed in microtitre wells. In the methods initially developed, biotinylated PCR products were captured on streptavidin-coated microtitre wells followed directly by time-resolved fluorescence detection of hapten molecules incorporated into the product during PCR. These methods could easily detect mRNA representative of one Ph$\sp1$-positive leukemic cell amidst 500 000 Ph$\sp1$-negative cells. Subsequently, assays with even higher sensitivities were developed which could identify the specific BCR-ABL mRNA. These methods involved hybridization of denatured PCR products to complementary probes specific for the BCR-ABL junction. In the final phase of this study, a quantitative assay for BCR-ABL mRNA based on the co-amplification of the target with an RNA internal standard (IS) was developed. A recombinant DNA fragment, synthesized through PCR, was used to create the RNA IS. The target and IS contained the same primer recognition sites and generated PCR products of identical size distinguishable only by hybridization to two specific probes. The ratio of the fluorescence values obtained for the target and IS were linearly related to the initial target RNA in the range of 30-10 000 Ph$\sp1$-positive leukemic cells. The high degree of sensitivity characteristic of the proposed assays is attributed to the successful combination of PCR with time-resolved fluorometry. Furthermore, the methods avoid time-consuming electrophoresis and Southern blot and eliminate the hazards associated with radioisotopic detection making them suitable for the routine clinical laboratory.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1996 .B685. Source: Dissertation Abstracts International, Volume: 59-08, Section: B, page: 3891. Adviser: Theodore K. Christopoulos. Thesis (Ph.D.)--University of Windsor (Canada), 1996.
Bortolin, Susan., "Polymerase chain reaction coupled to time-resolved fluorometry for the determination of chronic myelogenous leukemia-specificmRNA." (1996). Electronic Theses and Dissertations. 3121.