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Functional Genomics Part II.docx

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BIOL 3150

Functional Genomics Part II: Protein-DNAinteraction assays and the ENCODE (Chapter 11) • Study protein-DNAinteractions by: o EMSA(Electrophoretic mobility shift)Assay or Gel ShiftAssay  Used to identify which DNAsegment binds to a protein or protein complex  How: probe added to protein with/without antibody, then are ran on a polyacrylamide gel, any DNAprobe unbound will run faster and end up in band near bottom and any bounded DNA-protein complex will run much slower resulting in a band that is higher up on gel, any binding of antibody to protein-DNAcomplex will cause an even slower band and is known as a supershift o DNase I footprinting  Used to identify region of DNAoccupied by a protein complex  How: End labeled restriction fragments combined with nuclear extract and without, proteins bind to DNAwith nuclear extract, Limited DNase I digestion (cannot cut where protein), so will have gap where proteins bound (footprint), will know protein binding somewhere in this region  Can couple with NGS to identify DNase I hypersensitivity for entire genome (peak = greater accessibility to DNAregion), footprints correspond to transcription factor binding sites o Chromatin Immunoprecipitation (ChIP)  Used to identify protein-DNAinteractions  How: crosslink protein to DNAin living cells with formaldehyde, break open cells and shear DNA,Add pre blocked Protein G agarose beads, add primary antibody of interest, immunoprecipitate to enrich for fragments bound by protein of interest, reverse crosslinks and treat with proteinase K, detect and quantify precipitated DNAthrough PCR and hybridiza
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