In vitro analysis of DNA-protein interactions by proximity ligation.
Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.
Gustafsdottir
Sigrun M
SM
Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Dag Hammarskjöldsväg 20, SE-75185 Uppsala, Sweden. sigrun.gustafsdottir@genpat.uu.se
Schlingemann
Joerg
J
Rada-Iglesias
Alvaro
A
Schallmeiner
Edith
E
Kamali-Moghaddam
Masood
M
Wadelius
Claes
C
Landegren
Ulf
U
D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005821:Genetic Techniques; D008958:Models, Molecular; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction
D004268:DNA-Binding Proteins; D015335:Molecular Probes; D009841:Oligonucleotides; D004247:DNA