Enzyme digestion of entrapped single-DNA molecules in nanopores

The real-time digestion of entrapped single-DNA molecules by lambda-exonuclease in nanoporous alumina membranes was observed using an epifluorescence microscope. The alumina membrane provides pL (similar to 10(-12) L) containers for confining single-DNA molecules without immobilization. When one end of the DNA molecule was inserted into a nanopore, it was possible to monitor the digestion process outside, near and inside the pore, where the individual DNA molecules exhibited different characteristic digestion modes. The digestion rates calculated from the decrease in fluorescence intensity showed different values according to the location of the individual molecules. Entrapment rather than immobilization allows the DNA strand to be fully exposed to the enzyme and the reaction buffer. These results confirm that the enzymatic digestion of DNA molecules is affected by their three-dimensional (3D) environment. (C) 2011 Elsevier B.V. All rights reserved.