Single Molecule DNA Restriction Analysis in the Light Microscope

A combination of single molecule fluorescence intensity analysis and optical mapping is developed to identify individual fluorescently labeled DNA molecules on the basis of restriction patterns generated by different enzymes. Fluorescently labeled lambda-phage DNA molecules each bound to a polystyrene microsphere as a handle are held and moved by optical tweezers. The single DNA molecules are stretched by hydrodynamic flow. The restriction endonucleases Apa I, Sma I and EcoR I with one, three and five expected cutting sites on the lambda-phage DNA molecule are used for enzymatic digestion of the DNA molecules. The DNA restrictions are observed after microinjection of the enzyme into the flow towards the DNA molecule in real time (video frequency) on a microscope cover slide. The fluorescence intensity of the DNA fragments is measured and their length in terms of base pairs is calculated. Comparison with the length expected from sequence data reveals a single DNA fragment sensitivity.