Singly labeled smart probes for real-time monitoring of the kinetics of dNTP misincorporation and single nucleotide extension in DNA intra-molecular polymerization

In this paper, a simple and rapid method was developed for real-time monitoring of the kinetics of dNTP misincorporation and single nucleotide extension in DNA intra-molecular polymerization by using singly labeled fluorophore-oligonucleotide smart probes. The probes are designed with a self-complementary T-end and a sequence of stacked cytosines at the 5'-end, to which a fluorescein (FAM) is attached. When the DNA polymerase is introduced, it will bind to the 3'-end of the probe and catalyze the extension reaction, resulting in the formation of stacked guanines, which will instantly quench the fluorescence of FAM through photoelectron transfer. The method can accurately quantify the activity of the Klenow fragment of Escherichia coli DNA polymerase I with the exonuclease activity inactivated (KF-) in 3 min with a detection limit down to 3.7 pM, which is much faster and more sensitive than the existing technology in monitoring the polymerization in bulk reaction. Moreover, the smart probes could be used to determine the kinetics of dNTP misincorporation and single nucleotide extension by proper design of the sequence. The method is universally adaptive to any fluorescence spectrometer and offers a very convenient and cost-effective way for characterization of the fine kinetic procedures in DNA polymerization. (C) 2009 Elsevier B.V. All rights reserved.