Doctor of Philosophy (PhD)
The traditional format of electrophoresis, the slab gel, is quickly being replaced by capillary and microdevice platforms, which offer improvements in cost, resolution, speed, quantitation and automation in genetic analyses. These techniques also employ a variety of separation matrices while the slab gel is limited to agarose and polyacrylamide. The research presented here explores the use of these electrophoretic formats for the detection of single nucleotide mutations using two genetic models associated with human disease. Slab gel based heteroduplex analysis (HDA), a popular mutation scanning method, uses a specially designed universal heteroduplex generator (UHG) containing controlled variation to enhance the subtle conformational differences caused by single-base substitutions that are difficult to discriminate. Here, the slab gel based HDA-UHG method has been modified to capillary and microdevice formats for the quantitative and reproducible analysis of single-base substitutions in rpoB that give rise to the rifampin-resistant phenotype of Mycobacterium tuberculosis. The capillary method reduced analysis time from 2.5 hours to approximately 30 minutes. The microdevice further reduced analysis time to 6 minutes while maintaining efficiency and resolution. Both capillary and microdevice methods employed methyl cellulose as the sieving matrix at 0.3% and 0.75% (w/v) concentrations, respectively. In colorectal cancer, base substitutions in the K-ras gene occur early in development, are preserved throughout the course of tumor progression and thus, can be used as biomarkers for the diagnosis of early, curable tumors. The current detection scheme uses slab gel electrophoresis and a mutation specific method, multiplexed polymerase chain reaction/ligase detection reaction, to identify all 19 possible single-base substitution mutations at codons 12, 13 and 61. This technique was also adapted and optimized to the capillary and microdevice formats. This study evaluated capillary methods employing both cross-linked as well as entangled polymer matrices. The capillary methods ranged from 30 to 45 minutes in analysis time. The cross-linked capillary exhibited increased deterioration at longer electrokinetic injection times, while severe injection biases were observed in the entangled polymers evaluated. Initial microdevice experiments were possible in approximately 5 minutes using the entangled polymer matrices and have great potential in microdevice analysis for such mutations.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Thomas, Gloria, "Capillary and microdevice electrophoretic tools for genetic analyses: heteroduplex analysis for tuberculosis drug susceptibility and ligase detection reaction for colorectal cancer detection" (2002). LSU Doctoral Dissertations. 1613.