title: 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) (R(2) = 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.
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, China.
authors: Fengzhou
authors: F
authors: Xu
authors: Hui
authors: H
authors: Shi
authors: Xiaoxiao
authors: X
authors: He
authors: Kemin
authors: K
authors: Wang
authors: Xiaosheng
authors: X
authors: Ye
authors: Lv'an
authors: L
authors: Yan
authors: Shuyong
authors: S
authors: Wei
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D006108:Graphite / Q000737:chemistry*; D010087:Oxides / Q000737:chemistry*
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D010087:Oxides; D006108:Graphite; D004259:DNA-Directed DNA Polymerase