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œœœœœ@DNase I footprinting@œœœœœ



@DNase I footprinting is a method for determining the site of binding for a protein on a DNA sequence. In order to use this procedure, you must have: purified protein, the DNA sequence of the cloned DNA in question, and filter-binding data indicating the general region of binding (or at least good reason to believe the binding site is within a relatively small region). Before starting the actual DNase I footprinting, you need to isolate singley end-labeled probes of both strands containing the binding region, dissolved in ddH2O to 10,000 - 20,000 CPM/ul. These probes should be tested by DMS reactions (use 1/10 of the total amount of each probe - the rest of the DMS-treated probe is used for MW markers during the footprinting) for purity and to be sure the probes are the correct fragments.
@Once you have these probes, you must determine the correct amount of DNase I required for apprpriate partial digestion of EACH PROBE empirically by titration. With this information you are ready to begin DNase I footprint assays. Both the titration & footprinting are explained here.

  1. Reagents

    1. DNase I stock
      @1mg/ml RNase-free DNase I in TMK buffer
      @Store at -20C.

    2. TMK buffer
      50% glycerol
      50mM Tris pH8
      50mM KCl
      10mM MgCl2

    3. 2X footprint buffer
      100mM KCl (adjust to optimize binding)
      20mM MgCl2
      40mM Tris pH8
      0.2mM EDTA

    4. NTP mix
      3.3mM ATP
      3.3mM GTP
      3.3mM CTP

    5. STOP solution
      0.1M EDTA
      0.6M NH4OAc
      20ug/ml sssDNA

  2. Methods

    1. DNase I titration

      1. For each probe, mix (working on ice):
        440ul ddH2O
        50ul 5mg/ml BSA (nuclease-free)
        500ul 2X footprinting buffer
        50ul NTP mix
        10ul probe DNA
        Divide the mix into 10 reactions of 100ul each, in eppendorf tubes.

      2. Dilute 40ul stock DNase I in 116ul TMK (=256ug/ml DNase I). Do 8 1:2 serial dilutions of this 256ug/ml solution (75ul TMK + 75ul DNase I, mix, go to the next dilution).

      3. With all samples starting on ice, the experimental time course is :
        tube # 1 2 3 4 5 6 7 8 9 10
        --> 30C 0 1 2 3 4 5 6 7 8 9
        DNase(5ul) 10 11 12 13 14 15 16 17 18 19
        100uLSTOP->ice 12 13 14 15 16 17 18 19 20 21
        [DNase] used 0 1 2 4 8 16 32 64 128 256

      4. When all the samples are done, add 100ul phenol:chloroform to each & vortex. Spin 1-2 min and transfer the upper phases to tubes containing 600ul EtOH. Freeze the samples in a dry-ice:EtOH bath for 5 min, then spin for 5 min. Discard the supernatants and add 750ul ice-cold 70% EtOH to each tube, refreeze and spin as before.
      5. Resuspend each sample in 5ul sequencing dye and run a 6% sequencing gel. Run an appropriate distance for the size of the DNA used (bromophenol blue to bottom for 150bp, XC to bottom for 400bp). Dry & autoradiograph the gel (overnight exposure with screens at -70C).
      6. Choose the best partial digest for future use - you want even cleavage at all sizes (not faded at the top) with uncut DNA left over and the darkest partial bands.

    2. Footprinting

      1. Working on ice, mix the same reaction mix as before for each probe. Divide the mix into 10 tubes each of 100ul. Prepare dilutions of DNase I from the stock as before. You will use 5 different dilutions - the optimum and 2-fold dilutions up and down from this optimum (i.e. if 1:32 is best, use 1:128, 1:64, 1:32, 1:16, & 1:8) to make sure you get the best results.
      2. With all samples starting on ice, the time schedule is :
        tube # 1 2 3 4 5 6 7 8 9 10
        --> 30C 0 1 2 3 4 5 6 7 8 9
        DNase(5ul) 10 11 12 13 14 15 16 17 18 19
        100uLSTOP->ice 12 13 14 15 16 17 18 19 20 21
        [DNase] used 0.25 0.5 1x 2x 4x 0.25 0.5 1x 2x 4x
        relative to opt. @ @ @ @ @ @ @ @ @ @
        protein added? - - - - - + + + + +
        f required by protein concentration, more (up to about 10ul depending on what it's dissolved in) binding protein can be used.

      3. Phenol:chloroform extract, EtOH precipitate, 70% wash, dry, resuspend & run the 6% sequencing gel as before. Dry the gel and expose as indicated by experience from the DNase I titration.
      4. Repeat the footprinting using ddH2O in place of NTP mix.
      5. Examine the autoradiographs for gaps in the DNase I ladder found in the samples with added binding protein but not in the lanes without added RNAP.

JWB, unpublished
Craig & Nash 1984 Cell 39:707-719
Galas & Schmitz 1978 Nucl. Acids Res. 5(9):3157

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