Error-compensating kinetic method for enzymatic determination of DNAs.
We describe the adaptation and evaluation of an error-compensating method for kinetic determinations of deoxyribonucleic acids (DNAs). 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 model for two-component parallel first-order processes is fit to the decay curve to predict the total change in fluorescence expected if the process were monitored to equilibrium. The predicted change in fluorescence response varies linearly with DNA concentration with an intercept corresponding to 0.13 mg/L DNA. Results by the predictive method are 47-, 58-, and 250-fold less dependent on DNase activity, temperature, and ethidium bromide concentration, respectively, than are results for an initial-rate method utilizing the same data. Moreover, the predictive method yields a significantly wider linear range than the initial-rate method, and is much less affected by blank fluorescence and RNA interference than is an equilibrium method based on the reaction of DNA with ethidium bromide alone.
Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393.
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Pardue
D002138:Calibration; D004247:DNA / Q000032:analysis*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004996:Ethidium; D006801:Humans; D007700:Kinetics; D013696:Temperature
D004247:DNA; D003851:Deoxyribonucleases; D004996:Ethidium