Determination of protein-DNA sequence specificity by PCR-assisted binding-site selection

Binding-site selection is used to determine the target specificity of a sequence-specific DNA-binding protein. In this unit, a pool of random-sequence oligonucleotides is used as the source of potential binding sites. This pool is incubated with extract containing the DNA-binding protein of interest and the protein-DNA complexes are isolated by immunoprecipitation with an antibody specific for the protein under investigation. Unbound oligonucleotides are removed by gentle washing, and bound oligonucleotides are recovered, amplified by the polymerase chain reaction (PCR), and used as input DNA for a further round of binding, recovery, and amplification. After four rounds of selection, progress of the procedure is monitored by mobility shift analysis of the selected oligonucleotide pools. In the , individual binding sites are isolated from the appropriate complex on a mobility shift gel, cloned into plasmids, and examined by sequencing.