footprinting2.ppt

DNase footprinting DNase Footprinting assay is a technique used in molecular biology by which it is possible to determine the exact DNA sequence to which a particular DNA-binding protein (hormone-receptor complexes that bind to their hormone response elements, transcription factors that bind eukaryotic operators, enhancers, and silencers, the lac repressor that shuts down the?lac operon in E. coli) binds. It involves endonuclease treatment of an end labelled DNA fragment bound to a protein. Limited digestion produces fragments terminating everywhere except in the footprint region, which is protected from digestion. The technique using restriction endonuclease (or restriction enzymes) uses an enzyme that cuts double-stranded DNA. The enzyme, without damaging the bases, makes two incisions(切口） – one through each of the phosphate backbones of the double helix Provided the ends are complimentary, the chemical bonds that the enzymes sever （切断）can be reformed by other enzymes known as ligases, so that restriction fragments carved（刻） from different chromosomes or genes can be spliced together. To investigate DNA-protein interactions, the cleavage pattern of isolated DNA is compared with the cleavage pattern of DNA in the presence of the commonly accepted binding protein. If the protein binds the DNA, the binding site is protected against restriction enzyme cleavage and fewer cleavage sites are found. From the position of the cleavage sites absent in the assay containing the binding protein, the position and extension of the binding site can be deduced. First, a target DNA fragment about 100-300 bp in length is either PCR generated or cut from a vector and then uniquely labeled (at only one end) with a radioactive molecule, e.g. radioactive ATP, and incubated with protein (usually nuclear extract), followed by controlled digestion with deoxyribonuclease I (DNase I), an endonuclease digests nucleic acids starting in the middle of the strand (as opposed to an exonucl