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RESEARCH DIVISION Laboratory Manual

 


 

DNA-se I Footprinting

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).

Binding reaction:

Mix in a 50ul final volume:

0-25ul protein extract in HEMG (see below)
10ul 10% polyvinyl alcohol (store 4C)
1ul 1M Hepes pH 7.6 (pHed with KOH)
~2fmoles of end-labelled DNA probe (~20cps on minimonitor) competitor DNA (see below)
ddH2O to a final volume of 50ul

If the amount of protein used is less than 25ul, add 0.1M HEMG to make up the difference. Mix components gently and incubate ~10 minutes. Incubation on ice is usually required when dealing with relatively crude protein extracts which contain endogenous nucleases and/or phosphatases. With purified proteins the incubation temperature can be increased.

It is advisable to add non-specific competitor DNA, such as sonicated calf thymus DNA, synthetic poly d(I-C) or poly d(A-T) (Pharmacia) to the binding reaction. The optimal competitor concentration needs to be determined empirically, however 0.1-0.5ug of sonicated calf thymus DNA or 0.01 OD units of synthetic DNA are usually adequate.

DNAase I digestion:

To the binding reaction add 50ul Ca/Mg solution (10mM MgCl2, 5mM CaCl2) at room temperature and 1-10ul of DNAaseI solution (see below). Mix quickly and incubate at room temperature for 1 minute. Stop the reaction by adding 100ul of stop solution (0.2M NaCl, 20mM EDTA pH 8, 1% SDS, 0.25 mg/ml carrier RNA; store at room temperature) and vortexing immediately. Digestion of three samples can be carried out simultaneously with a bit of practice.

Samples are then extracted with 200ul of 1:1 phenol:chlorophorm and precipitated with 1ml of 100% ethanol. Pellet is washed with 70% ethanol, dried and resuspended in 6ul of formamide dye. Run on 6-8% sequencing gel.

HEMG:
0.1M KCl
25 mM Hepes pH 7.6 (pHed with KOH)
0.1 mM EDTA pH 8
12.5mM MgCl2
10% glycerol
1mM DTT (fresh)

 

DNAase I:

Make a 2.5mg/ml DNAaseI (Worthington or Cooper Biomedical, bovine pancreas, DPFF, 2000U/mg) solution in ddH2O and freeze 2-5ul aliquot’s; keep at -70C. Make a fresh dilution of this stock solution in ice cold ddH2O and keep on ice. For the no-protein control use ~1ul of a 1/1000 dilution. For crude protein extracts the amount and dilution of DNAaseI needs to be determined; 5ul of a 1/100 dilution are usually adequate.

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998