Date of Award
Doctor of Philosophy
Edward S. Yeung
The purpose of the Human Genome Project is outlined followed by a discussion of electrophoresis in both slab gels and capillaries and its application to deoxyribonucleic acid (DNA). Techniques used to modify electroosmotic flow in capillaries are also addressed. Several separation and detection schemes for DNA via gel and capillary electrophoresis are described. Emphasis is placed on the elucidation of DNA fragment size in real time as well as shortening separation times to approximate real time monitoring;First, the migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis are possible throughout the separation via this detection scheme. The use of absorption removes the need for staining or radioisotope labeling thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without de-staining. The optimization of several parameters considerably reduces total analysis time. DNA from 2 kb to 850 kb can be separated in 3 hours on a 7 cm gel with interactive control of the pulse time, which is 10 times faster than the use of a constant field program;Finally, the separation of [phi]X174RF DNA-HaeIII fragments is studied in a 0.5% methyl cellulose polymer solution as a function of temperature and applied voltage. The migration times decrease with both increasing temperature and increasing field strength, as expected. However, the relative migration rates of the fragments (selectivities) do not change with temperature but are affected by the applied field. A clear transition from the Ogston separation mechanism to the reptation separation mechanism is observed. Conditions were established for the separation of the 271/281 bp fragments, even without the addition of intercalating agents. At 700 V/cm and 20°C, all fragments are separated in less than 4 minutes with an average plate number of 2.5 million per meter.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
David Arthur McGregor
McGregor, David Arthur, "Optimization of separation and detection schemes for DNA with pulsed field slab gel and capillary electrophoresis " (1993). Retrospective Theses and Dissertations. 10251.