Summary: DNase I Footprinting assay is a method of studying DNA-protein interaction and identifying the DNA sequence to which a protein binds. First, a target DNA fragment about 100-300 bp in length is either PCR generated or cut from a vector and then uniquely labeled (at only one end) and incubated with protein (usually nuclear extract), followed by controlled digestion with DNase I which cut the probe randomly but only once. The digested DNA is recovered from the reaction and resolved on a polyacrylamide gel along with G+A chemical sequencing reaction which uses the same probe as template. The regions bound by proteins will be protected from DNase I digestion and will be shown as a blank area on the gel track, while the exact protein-bound sequence can be read out by comparing the location of the blank with the sequencing reaction.
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- DNase I Footprinting (BioProtocol)
An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once. Digestion products are then resolved by electrophoresis. Comparison of the DNase I digestion pattern in the presence or absence of protein will allow the identification of a footprint (protected region).
http://www.bio.com/protocolstools/protocol.jhtml?i...
Added: Tue Mar 23 2004, Hits: 4332, Reviews: 0 Write review Cached - Footprinting Analysis
(The Morimoto Lab, Northwestern University)
This protocol describes multiple footprinting methods such as DNase I, Fe-EDTA OR hydroxyl radical, methidiumpropyl-EDTA (MPE), DMS protection. It also offers detailed methods for probe preparation.
http://www.biochem.northwestern.edu/morimoto/resea...
Added: Mon Mar 29 2004, Hits: 3790, Reviews: 0 Write review - Preparation of End-Labeled DNA Probes by Conventional Kinase for DNA Footprinting Analysis
Preparing probe for DNase I footprinting assay can be very tricky. There are several strategies for designing a footprinting probe. This protocol gives very detailed guide from initial restriction digestion, labeling, to final purification.
http://www.bio.com/protocolstools/protocol.jhtml?i...
Added: Tue Mar 23 2004, Hits: 1294, Reviews: 0 Write review Cached - DMS Chemical Footprinting of RNA (Laederach Lab)
Dimethyl Sulfate is used as a chemical probe by methylation of A's and C's. Primer extension with a cy'5 labeled primer and reverse transcription allow us to determine secondary structures of RNA.
Added: Thu Dec 17 2009, Reviews: 0 Write review -
DNA Footprinting
(Kimball's Biology Pages)
If you want to know what footprinting is, here is the right place for you to start with. This page gives the principles behind footprinting and procedures as well. Along with the text, it also provides a illustration of this method.
http://users.rcn.com/jkimball.ma.ultranet/BiologyP...
Added: Tue Mar 23 2004, Hits: 2464, Reviews: 0 Write review Cached -
DNA-se I Footprinting
(Bowtell Lab Manual, Peter MacCallum Cancer Institute)
This technique exploits the fact that a protein bound to a specific DNA sequence will interfere with the digestion of that region by the endonuclease DNAaseI. In summary, an end-labelled DNA probe is incubated with a protein extract or a purified DNA-binding factor. The unprotected DNA is then partially digested with DNAaseI such that on average every DNA molecule is cut once. Digestion products are then resolved by electrophoresis. Comparison of the DNAaseI digestion pattern in the presence and absence of protein will allow the identification of a footprint (protected region) or hypersensitive sites (sites at which digestion is enhanced due to protein binding).
http://www.pmci.unimelb.edu.au/core_facilities/man...
Added: Tue May 14 2002, Hits: 483, Reviews: 0 Write review Cached -
DNase I Footprinting
(Mike A. Dyer)
http://axon.med.harvard.edu/~cepko/protocol/mike/F...
Added: Tue May 14 2002, Hits: 940, Reviews: 0 Write review Cached -
DNase I Footprinting
(Bowtell Lab)
http://grimwade.biochem.unimelb.edu.au/bowtell/mol...
Added: Tue May 14 2002, Hits: 326, Reviews: 0 Write review Cached -
DNase I Footprinting
(Tsukiyama Lab, Fred Hutchinson Cancer Research Center)
http://labs.fhcrc.org/tsukiyama/protocols/dnase_i_...
Added: Tue Mar 03 2009, Hits: 1720, Reviews: 0 Write review - Preparation of G+A Marker ( Protocol Online)
Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing reaction and works fine.
Added: Tue May 14 2002, Reviews: 0 Write review