Error-compensating kinetic method for enzymatic determination of DNA
Abstract
This thesis describes the adaptation and evaluation of an error-compensating method for kinetic determinations of DNA. The DNA is first reacted with ethidium bromide to produce a fluorescent intercalation complex. Subsequent treatment of the complex with DNase catalyzes hydrolysis of the DNA, causing a time-dependent decrease in fluorescence, which is monitored. A suitable model is fit to the decay curve to predict the total change in fluorescence expected if the process were monitored to equilibrium. The results are compared to the commonly used equilibrium and initial-rate methods. Both the determination of DNA in homogeneous solution and in agarose gel are studied. For experiments in solution, a model for two-component parallel first-order processes is fit to the response curves. Results by the predictive option are 58-, 250-, and 47-fold less dependent on temperature, ethidium bromide concentration and DNase activity, respectively, than are results for the initial-rate method utilizing the same data. The predictive method also yields a significantly wider linear range than the initial-rate method and offers the automatic background-correction advantage relative to the equilibrium option. For experiments in gels, a single size DNA is used for the initial study. The experimental procedures involve gel electrophoresis followed by DNase treatment of the agarose gel containing the single DNA band. The disappearance of the DNA band is monitored using a charge-coupled device camera. A Michaelis-Menten model is fit to the corrected response curves. Results obtained using the predictive and equilibrium options are linear over two decades of DNA (0-120 ng) with 3 to 6% imprecisions. For variable dependency studies, results by the predictive option are 26-, 14-, and 387-fold less affected by ethidium bromide concentration, DNase activities and gel concentration, respectively, than are results for the initial-rate method. A DNA sample containing multiple fragments is later used to demonstrate the applicability of this method for the determination of DNA.
Degree
Ph.D.
Advisors
Pardue, Purdue University.
Subject Area
Analytical chemistry|Biochemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.