A sensitive method for the determination of protein-DNA binding specificities.

AUTOR(ES)

Pollock, R

RESUMO

We describe a sensitive and rapid method for determination of the sequence specificity of DNA binding proteins. The method allows recovery of specific sites using the small amounts of protein present in crude cell extracts or produced by cell-free translation reactions. Extract proteins are incubated with a pool of random sequence oligonucleotides, complexes purified by immunoprecipitation, and bound DNA amplified by the Polymerase Chain Reaction (PCR). This DNA is then used in further rounds of binding, immunoprecipitation, and amplification, until specific binding is detectable. With the transcription factor SRF as a model system, we demonstrate that authentic high affinity binding sites are recovered, and show that epitope tagging can be used to allow recovery of sites when specific antibodies are unavailable. We also show that specific sites bound by the Fos protein, which binds DNA with high affinity only when complexed with other polypeptides, are easily recovered by this technique.

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