Highly rugged kinetic method for the enzymatic determination of DNA in agarose gel with array detection using a charge coupled device

This paper describes the adaptation and evaluation of an error-compensating method for kinetic determinations of DNA. A single size DNA is used for the initial study. The experimental procedures involve gel electrophoresis with ethidium bromide staining followed by DNase treatment of the agarose gel containing the single DNA band. The time-dependent decrease in fluorescence of the DNA band is monitored using a charge-coupled device camera. A Michaelis-Menten model is fitted to background—and flat-field-corrected response curves to predict the total change in fluorescence expected if the process were monitored to equilibrium. The results are compared to equilibrium and initial-rate methods. Results obtained using the predictive and equilibrium options are linear over 120-fold amounts of DNA (1–120 ng) with 3–6% imprecisions. The equilibrium and predictive-kinetic options are at least an order of magnitude more rugged to changes in ethidium bromide concentration, DNase activity and gel concentration than the initial-rate option. A DNA sample containing multiple fragments was used to evaluate the applicability of this method for more realistic situations.