A Dual-Beam Optical Microscope for Observation and Cleavage of Single DNA Molecules

Abstract

An integrated epifocal and evanescent-wave optical microscope has been developed for real-time observation and bond cleavage studies of single DNA molecules. Large genomic DNA is stretched in a laminar flow stream and is immobilized on a polylysine-coated glass surface by strong electrostatic interactions. Unlike previous single-molecule dynamics and single-enzyme studies, this work takes advantage of the elastic nature of double-stranded DNA and measures DNA relaxation events that are triggered by two phosphodiester (P-O) bond breaks. The ability to follow chemical reactions on individual DNA molecules opens new possibilities for DNA mapping and for studying DNA-protein interactions.