title: A polyplex qPCR-based binding assay for protein-DNA interactions.
abstract: The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.
authors.affiliation: School of Pharmacy and Molecular Sciences, James Cook University, Townsville, QLD, Australia.
authors.forename: Morgane J J
authors.initials: MJ
authors.lastname: Moreau
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
mesh_terms: D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D047468:Immunoprecipitation; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C489236:tus protein, E coli; D049452:Green Fluorescent Proteins