Fluorescence-Based DNA Polymerase Assay

Abstract

The standard procedure for assaying DNA polymerase activity involves use of DNase-treated DNA (“activated DNA”) as a primer/template and incorporation of radioactive nucleotides into DNA (1). Measurement of acidprecipitable radioactivity relative to total radioactivity allows calculation of the amount of nucleotides incorporated and the number of enzyme units present (one unit of DNA polymerase activity is conventionally defined as the amount of enzyme that will incorporate 10 nmol of nucleotides during a 30-min incubation). However, use of radioactivity is restricted and discouraged in many laboratories, and there is a general trend away from radioactivity-based techniques. For DNA polymerases, a commercial assay based on ELISA detection of digoxigenin-labeled nucleotides incorporated into newly made DNA is available (Roche Molecular Biochemicals, Cat. No. 1669885). Also a fluorescence-based assay for DNA polymerase holoenzyme, based on the specific reaction of the dye PicoGreen with double-stranded DNA, has been published (2). We have modified this procedure to make it suitable for less processive and complex DNA polymerase systems. The method described here allows quantitation of polymerase activity in preparations of family A and B polymerases at reaction temperatures between 37 and 72°C, exemplified by the Klenow fragment of DNA polymerase I from Escherichia coli, Taq DNA polymerase, and Vent polymerase.