Exonuclease III footprinting on immobilized DNA templates

Abstract

DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from degradation by an enzyme or chemical reagent. Exonuclease III is a suitable probe to analyze the boundaries of a protein when it is necessary to eliminate any excess unbound DNA from the reaction to avoid background problems. In combination with biotin-labeled DNA that is bound to streptavidin-coated magnetic particles, information on the precise position of a DNA bound protein is available within a few hours. The position of the archaeal RNA polymerase at different stages of transcription in the Pyrococcus furiosus in vitro transcription system was analyzed by this method. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.