Optical mapping of DNA polymerase I action and products

Single molecule approaches to the characterization of biochemical systems o ffer an intrinsically simple and direct approach to address difficult, prev iously unyielding problems. Optically based approaches have recently been u sed to construct high resolution, ordered restriction maps from a variety o f clone types. Advancements in surface technologies have enabled the reliab le elongation and fixation of large DNA molecules onto specially derivatize d substrates with retention of biochemical accessibility. In this study, th e addition of fluorescently labeled nucleotides to surface-mounted DNA mole cules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nic k translation and containing only a few fluorochromes are readily imaged. T hese novel results suggest that surface-bound molecules may serve as a subs trate for a broad range of enzymatic actions, and may offer new routes to a nalysis when coupled to advanced imaging techniques. (C) 1999 Academic Pres s.