Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Lloyd Wolfinbarger, Jr.
Mark S. Elliot
In Part I of this dissertation, empirical equations for predicting DNA mobility during agarose gel electrophoresis (AGE) from voltage gradient are developed from the data of McDonnel (36) for electrophoresis in a 1.6% agarose gel. These equations represented the data well for DNA between 2 and 10 kilobase pairs (KBp) in length. A computer program, called GELSIM, which incorporates these equations is described in Part II. GELSIM was designed to allow researchers to analyze electrophoresis data by predicting the effect on DNA migration of altering the voltage of electrophoresis. In this way, electrophoretic banding patterns produced using different voltages could be compared. GELSIM was tested by comparing its predicted banding patterns for a DNA standard with those actually obtained in the lab. The patterns matched well for voltages below 5 volts per centimeter of electrode separation, the maximum recommended electrophoresis voltage gradient (34).
In Part III, a characterization of the voltage gradient using an electrode grid to measure local voltages, it was found that the voltage gradient is linear from anode to cathode and invariant from lane to lane in the gel. It was also determined that the gradient within the gel was significantly lower than that predicted by simply dividing the electrophoresis voltage by the electrode separation, the usual practice.
Part IV of this dissertation details a measurement of local voltage gradient during electrophoresis over very short intervals of time. The apparatus used to make these measurements consisted of an electrode array hooked via an analog to digital converter to a computer. It was found that the local voltages were not constant, as expected, but fluctuated in a roughly sinusoidal manner by as much as 10% of their average value. The frequency of the fluctuations was 60 Hz. The source of the fluctuations was determined to be the DC power supply used during the electrophoresis and the form of the fluctuations, was found to vary from power supply to power supply. It was concluded that these fluctuations were a general feature of AGE and likely to be of theoretical as well as practical importance in electrophoretic separation.
"The Role of the Voltage Gradient in the Agarose Gel Electrophoresis of DNA"
(1990). Doctor of Philosophy (PhD), Dissertation, Chemistry and Biochemistry, Old Dominion University, DOI: 10.25777/mkz8-7552