Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA.

ABSTRACT

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.