Techniques to Measure Nucleic Acid-Protein Binding and Specificity: Nuclear Extract Preparations, DNase I Footprinting, and Mobility Shift Assays

DNA-protein interactions are the basis for the molecular mechanisms responsible for nucleic-acid replication, gene transcription, recombination, viral integration, and gene regulation in both normal and pathophysiological conditions. Initially, deletional analysis coupled with reporter gene assays are used to roughly map the locations of regulatory regions involved in gene expression. Relevant cis-regulatory elements may also be detected as areas of DNase I hypersensitivity in native chromatin. DNase I footprinting analysis is subsequently used to precisely locate sites of DNA-protein interactions within the identified regulatory regions. The proteins that interact with these regulatory regions can be detected and isolated using various modifications of the mobility shift assay. The protocols described below have been successfully used in our laboratory to analyze the regulatory regions of several genes (1 –6 ).