DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding Specificity

Abstract

INTRODUCTIONKnowledge of the DNA-binding specificity of a transcription factor aids in understanding the function of that factor in the regulation of gene transcription. One popular method of identifying the genomic DNA sites bound by a given protein in vivo is the Chromatin ImmunoPrecipitation with microarray analysis (ChIP-chip) technique. However, this method reveals a binding pattern influenced by in vivo phenomena that may mask the actual DNA-binding specificity of the factor, such as chromatin effects and competitive or cooperative protein-protein interactions. ChIP-chip also requires adequate expression of the protein in the cell type chosen to create the extract used for immunoprecipitation. DNA Immunoprecipitation (DIP) is an alternative technique that allows one to take advantage of the favorable properties of both in vivo and traditional in vitro techniques (e.g., electromobility shift assay [EMSA] and binding site selection [SELEX]). DIP utilizes naked genomic DNA as a binding substrate for one or more purified recombinant proteins. Because genomic DNA is used as a template in DIP reactions, the results are directly comparable to ChIP-chip or ChIP-seq data. DIP can be carried out in the presence of cofactors such as heterodimer partners, competitors, or small molecule binding inhibitors. After DNA is isolated by DIP, it is most efficiently detected using a highly parallel genomic technique such as a DNA microarray (DIP-chip) or high-throughput sequencing (DIP-seq). In this protocol, we describe a DIP of a yeast (Saccharomyces cerevisiae) protein with yeast genomic DNA.