Nonradiochemical DNase I footprinting by capillary electrophoresis

A new application for DNase I footprinting using capillary electrophoresis (CE) has been developed in order to decrease analysis time and to eliminate the use of radiochemicals. An additional advantage of the new method over the traditional radioactive methods is that the DNA probe can be labeled on both ends with different fluorescein dyes. This provides an internal check of the identification of protein-binding sites on DNA, because the binding region can be observed from both DNA strands. The initial parameters for t he CE method were developed using the Promega Core Footprinting Kit for ana lysis of AP-2 binding sites in the SV40 enhancer sequence. After optimizati on of the method, the protocol was found to be effective for footprint anal ysis of the immediate upstream region (bases -1 to -370) of the rat glutath ione peroxidase (GPX) and it permitted identification of a previously unkno wn binding site in the upstream sequence of the GPX gene.