Doctoral Dissertations
Date of Award
12-1995
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Chemistry
Major Professor
Michael J. Sepaniak
Abstract
The development of DNA fingerprinting, new therapeutic agents, £ind gene cloning has had a dramatic impact on the health and welfare of the human population. The key to their success lies hidden within the genetic code (DNA). The ability to rapidly and reliably analyze DNA is essential to the continued advancement of these promising technologies. Unfortunately, conventional analytical methodologies require large expenditures of time and effort to obtain useful information. Thus, there is a desire to decrease the overall analysis time. In this work, the development of capillary electrophoresis (CE) methodologies to decrease the overall DNA analysis time is undertaken by addressing detection, separation and instrumental concerns. On-column complexation using intercalation dyes with laser fluorometric detection provides sensitive detection of DNA restriction fragments. This is accomplished by the addition of the dye to CE running buffer and employing an inexpensive He-Ne laser for excitation of the DNA-dye complex. Detection limits in the low-mid-femtogram range are possible with a linear dynamic range of nearly 2 orders of magnitude. Soluble polymers provide excellent size discrimination of DNA restriction fragments when used as CE running buffer additives. Fundamental investigations were undertaken to determine the feasibility of performing high speed separations of DNA restriction fragment digests using size-selective CE. Good resolution and short analysis times (120 seconds) of the 11 fragments in a ΦX-174 Hea III digest with a size range of 70-1,300 bp were achieved using a 100,000 MW methyl cellulose at a field strength of -1,000 V/cm. Comparable separation times were achieved at moderate field strengths by adjusting the effective column length during the course of the separation process. Complete resolution of the aforementioned sample is accomplished in approximately 155 seconds with an effective column length of 15 cm using a field of -500 V/cm. Modified CE instrumentation capable of scanning the entire column length can be utilized in a developmental mode to further reduce the separation time. Fundamental investigations of band migration and dispersion in CE were undertaken using this instrumentation.
Recommended Citation
Clark, Brian Keith, "DNA restriction fragment analysis using size-selective capillary electrophoresis with laser induced fluorescence detection. " PhD diss., University of Tennessee, 1995.
https://trace.tennessee.edu/utk_graddiss/9960