What is dna footfrinting?

• Dna  Footprinting -
• Definition- It is a method of studying DNA-protein interaction and identifying the DNA sequence to which a protein binds.
• Function-  It measures the ability of a protein to protect a radiolabelled DNA fragment against digestion by DNase-I (endonuclease) with little sequence specificity that will cut almost anywhere within a DNA molecule.
• Procedure -
• A DNA fragment  contain sequences recognized by a DNA-binding protein is isolated and radio labeled at one end of one strand.
• Once the DNA fragments are labeled, they are mixed in a test tube with a DNA-binding protein and DNAse-I.
• In a separate test tube, same labeled DNA with DNAse is mixed without the DNA-binding protein. This is for comparison.
• DNAse is a cleavage agent which cuts the DNA in both samples. No cuts are made in the area where RNA polymerase has bound. It has protected the DNA.
• Only labeled strands are detected in next step.
• The free  DNA ( without protein )and just enough DNase 1 to cut on avg.once per molecule generates a population of fragments of all possible sizes that apper as a continuous ladder .
• Separation of fragments is done by polyacrylamide gel electrophoresis, and visualized by autoradiogram.
• Result-   The position of the footprint reveals where on the DNA molecule the protein has bound.

DNase -I  footprinting  assay-protein having specific binding site on DNA

Image source credit -http://www.nationaldiagnostics.com/images/2_1_9a.gif

MCQs.
1.Transcription initiation sites can be determined by-
(a) Foot Printing
(b) Northern Blotting
(c) Primer extension
(d) Nick translation
Ans.a

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