Doctoral Dissertations

Date of Award

12-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael J. Sepaniak

Committee Members

Cynthia B. Peterson, Spiro Alexandratos

Abstract

Capillary electrophoresis (CE) is a powerful analytical tool that has been applied to the separation of a variety of biomolecules including DNA. Recently, CE has found its way into the life sciences laboratory as a means to investigate analyses performed by the more traditional biochemical methods. The unique characteristics of CE (speed, efficiency (resolving power) and small sample requirements) are exploited for the investigation of DNA and protein molecules. Increasing pressure to extract specific information from DNA analysis in a timely fashion creates a burden on the development of effective experimental approaches. In this light, the development of computational methods for the optimization of DNA fragment separations based on content of sample and empirically derived relationships between fragment size, soluble polymer concentration and column length was undertaken. These models allowed for the optimal separation performance of all fragments in the sample. The development of a capillary-based assay for the investigation of protein-DNA interactions was also explored. Resolution of DNA from the protein-DNA complex is dependent upon buffer composition and pH. The selectively of these bio-specific interactions in demonstrated in the presence of interfering proteins. The diagnostic use of CE can further be realized in the temporal analysis of DNA restriction enzyme digests. Demonstrated is the ability to follow the progression of a digest (i.e., the creation and diminishing of fragment peaks) and to determine partial digest conditions for the generation of intermediate fragments of interest.

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