Fluorescent Marker for Direct Observation of Binding to Specific dsDNA Sequences

We have created a fluorescent marker that enables the direct, prolonged observation of binding to specific sequences on single molecules of double-stranded (ds) DNA using fluorescence microscopy. A mutant EcoRI restriction endonuclease (K249C) is selectively biotinylated and then incubated with dsDNA and Neutravidin-coated polystyrene spheres. The marker-laden DNA is fluorescently stained and subsequently stretched on slides so that the positions of the probes along the DNA can be measured. We provide evidence of the probe's ability to bind to its target sequence with high specificity using both bulk experiments and single-molecule methods.