ERROR-COMPENSATING KINETIC METHOD FOR ENZYMATIC DETERMINATION OF DNAS

We describe the adaptation and evaluation of an error-compensating met hod for kinetic determinations of deoxyribonucleic acids (DNAs). The D NA is first reacted with ethidium bromide to produce a fluorescent int ercalation complex. Subsequent treatment of the complex with DNase cat alyzes hydrolysis of the DNA, causing a time-dependent decrease in flu orescence, which is monitored. A model for two-component parallel firs t-order processes is fit to the decay curve to predict the total chang e in fluorescence expected if the process were monitored to equilibriu m. The predicted change in fluorescence response varies linearly with DNA concentration with an intercept corresponding to 0.13 mg/L DNA. Re sults by the predictive method are 47-, 58-, and 250-fold less depende nt on DNase activity, temperature, and ethidium bromide concentration, respectively, than are results for an initial-rate method utilizing t he same data. Moreover, the predictive method yields a significantly w ider linear range than the initial-rate method, and is much less affec ted by blank fluorescence and RNA interference than is an equilibrium method based on the reaction of DNA with ethidium bromide alone.