Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions.
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.
Stengel
Gudrun
G
Max-Planck-Institute for Polymer Research Ackermannweg 10, D-55128 Mainz, Germany. stengel@scripps.edu
Knoll
Wolfgang
W
D020137:Base Pair Mismatch; D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers; D005456:Fluorescent Dyes; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009711:Nucleotides / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*
D002232:Carbocyanines; D017931:DNA Primers; D005456:Fluorescent Dyes; D009711:Nucleotides; C085321:cyanine dye 5; D004247:DNA; D004256:DNA Polymerase I