Real-Time Fluorescence Assays to Monitor DNA Polymerase Activities

Abstract

We report fluorescence-based methods to study the polymerase and the exonuclease activities of thermostable DNA polymerases in real time. The methods are based on the use of a fluorescent reporter which become fluorescent upon binding to double-stranded DNA (method #1), the use of a quencher reporter strand displacement from a tripartite substrate (method #2) or the use of fluorescence fading of a nucleic acid stain concomitantly to DNA degradation (method #3). These methods are compatible with standard spectrofluorimeters, plate-readers or Real-Time PCR instruments. Here, we follow the efficiency of primer extension and degradation reactions by Pyrococcus abyssi thermostable family B DNA polymerases at 55°C. In addition, the size of extended products is systematically examined by gel electrophoresis followed by fluorescence visualization.These real-time methods are very sensitive, quantitative, and well suited for the screening of extension and exonuclease activity by DNA polymerases. Moreover, these assays could be used to characterize other DNA enzymes like helicases or nucleases.Based on operational properties of different DNA pols, these results could raise specific features with possible evolutionary scenario for the origin of families DNA polymerases.