A facile graphene oxide-based DNA polymerase assay

Abstract

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo⁻ (KF⁻) as a model. It was confirmed that, after optimization of detection conditions, KF⁻ activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL⁻¹ and a good linear correlation between 0.05–2.5 U mL⁻¹ (R² = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF⁻ activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.