DNAase footprinting

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A technique for determining the sequence of a DNA segment to which a DNA-binding protein binds. In this technique, a double-stranded DNA fragment is radioactively labelled at the 5′ end, partially digested with DNAase (q.v.) in the presence and absence of the binding protein, and the resulting fragments compared by electrophoresis (q.v.) and autoradiography (q.v.) on a gel that also runs in parallel the reaction products of a sequencing reaction performed on the unprotected sample of DNA. This produces an autoradiograph with ladders of oligonucleotides of varying lengths, increasing in single-nucleotide increments. The DNA region covered by the binding protein is protected from DNAase degradation and appears as a gap, or a footprint, that is missing from the sample lacking the protective protein. The footprint-containing ladder aligned with DNA sequencing ladders then identifies the exact sequence of bases in the footprint. See dimethyl sulfate protection, DNAase protection, DNA sequencing techniques.

Subjects: Genetics and Genomics.

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