Preparation of DNAase I Footprint Probes
Preparation of DNAase I Footprint Probes
DNA enzyme I footprinting analysis is an effective method for detecting and characterizing the sequence-specific binding ability of transcription factors to DNA (Galas and Schmitz, 197S). This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.
Operation method
Preparation of DNAase I Footprint Probes
Materials and Instruments
TE Bovine Intestinal Alkaline Phosphatase Buffer T4 Polynucleotide Kinase Buffer TBE Spiking Buffer TBE Plasmid DNA Suitable Restriction Enzyme Powder Chloroform Chloroform Isoamyl Alcohol (3-methyl-1-butanol) Ethanol Bovine Intestinal Alkaline Phosphatase [γ-32P]ATP T4 Polynucleotide Kinase Ammonium acetate Sodium acetate Acrylamide Bis-acrylamide Ammonium persulfate TEMED Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SpeedVac Rotary Concentrator
Plasmid DNA (with recognition sites for sequence-specific DNA binding factors to be isolated)
Suitable restriction enzymes
Powder/chloroform (1:1;v/v)
Chloroform/isopentanol (3-methyl-1-butanol) (24:1;v/v)
Ethanol (100% and 75%;v/v)
Bovine intestinal alkaline phosphatase (BoehringtrMannheimCorp. 713023)
[ γ-32P ]ATP (150uCi/ul; 7000Ci/mmol; 21pmol/ul)
T4 polynucleotide kinase (10U/ul) (Stratagene)
Ammonium acetate (2.5mol/L)
Sodium acetate (3mol/L)
SpeedVac Rotary Concentrator (Savant Instruments, Inc.)
Acrylamide
Bisacrylamide
Ammonium persulfate
TEMED (N,N,N',N'-tetramethylethylenediamine)
Reagents
TE
Bovine intestinal alkaline phosphatase buffer (10x)
T4 Polynucleotide Kinase Buffer (10x)
TBE Sampling Buffer (5x)
TBE(10x)
(For recipes, see "Reagent Preparation" pp.131-138.)
Operating Procedures
Plasmid DNA
The plasmid DNA used to prepare the DNAase I Footprint Probes must be of high quality (CsCl purified) and must be low in RNA contaminants, which can inhibit the kinase reaction. We generally use a two-step CsCl gradient to purify the DNA.
First restriction enzyme digestion
The purpose of the first restriction digest is to form the 5' protruding end. The position of the cleavage site should be within 50~200bp from the factor binding site.
1) Incubate plasmid DNA (50ug amount is convenient) with the first restriction enzyme and monitor the reaction by agarose gel electrophoresis to see if the reaction is complete.
2) DNA was extracted once with 1:1 phenol/chloroform and once with 24:1 chloroform/isoamyl alcohol.
3) DNA was precipitated with ethanol, dissolved in TE to a final concentration of 1ug/ul.
Remove the 5' end of the smectic acid
1) Add 1U of bovine alkaline phosphatase per μg of DNA [e.g., to dephosphorylate 50ug of DNA, add 50U of phosphatase to a total volume of 500ul (50U=50ul)] Incubate at 37°C for 2 h. Incubate for 2 hours.
2) DNA was extracted once with phenol, once with 1:1 phenol/chloroform and once with 24:1 chloroform/isoamyl alcohol.
3) Precipitate the DNA with ethanol, dissolve in TE to a final concentration of 1ug/ul.
Note; A convenient way to store large amounts (≥50ug) of DNA after digesting and dephosphorylating is to store it as a reservoir and label small amounts (typically 5ug) of DNA for backup when needed.
Labeling the 5' end
1) Add 5ul of digested and dephosphorylated DNA (1ug/ul; 5ug total; 5pmol 5' end) to 11ul of aqueous solution containing 2ul of 10xT4 Polynucleotide Kinase Buffer and shake to mix.
2) To this mixture, add 1ul of [ γ-32P ]ATP (the molar ratio of labeled ATP to the 5' end of DNA should be 2~4), and then add 1ul of T4 polynucleotide kinase (this kinase is in excess), the final volume of the reaction is 20ul.
3)Incubate at 37°C for 1~2 h.
4) Add 200ul of 2.5 ml/L ammonium acetate and mix with shaking. Centrifuge the solution in a microcentrifuge for 0.5s to bring the solution back to the bottom of the tube.
5) Heat at 70°C for 15 min to inactivate the kinase. Cool on ice.
6) Add 660ul of 100% ethanol, mix upside down and centrifuge for 15 min to precipitate the DNA. remove the supernatant with a pipette.
Note: Be very careful not to aspirate the DNA precipitate. Make sure that the precipitate has an appropriate level of radioactivity.
7) Add 100ul of TE, shake to mix and dissolve DNA.
8) Add 100ul of 1:1 phenol/chloroform, mix on shaker for 1 min, centrifuge for 5 min to separate phases.
9) Transfer the upper layer to a new tube, add 10ul 3 ml/L sodium acetate, shake and mix.
10) Add 300ul of 100% ethanol and mix upside down. Centrifuge for 15 min to precipitate the DNA and remove the supernatant with a pipette.
Note: Be very careful not to aspirate the DNA precipitate. Make sure that the precipitate has an appropriate level of radioactivity.
11) Add 800ul of 75% ethanol and mix upside down. Centrifuge for 15 min to precipitate the DNA and remove the supernatant with a pipette.
Note: Be extremely careful not to aspirate the DNA precipitate and confirm that the precipitate has an appropriate level of radioactivity.
12) Dry the precipitate using a SpeedVac rotary concentrator.
13) Dissolve DNA in 10ulTE.
Second restriction digest
The purpose of the second restriction digest is to produce a 200-800bp labeled DNA fragment.
1) Incubate the probe DNA with the second restriction enzyme. In order to ensure that this reaction is complete, it is recommended that a large excess of enzyme be used if possible, e.g., 50~100U of restriction enzyme for 5~10ug of DNA.
Note: Attention should be paid to the phosphatase activity of the restriction enzyme. In general, it is better to use inexpensive enzymes such as EcoRⅠ and HindⅢ. It is convenient to perform the reaction in 80ul volume. If necessary, the progress of the reaction can be monitored by agarose gel electrophoresis.
2) Extract the DNA with 1:1 phenol/chloroform, then add 20ul of 5xTBE Sampling Buffer to the 80ul of DNA, and add the sample directly to a 5% polyacrylamide (non-denaturing) gel.
Polyacrylamide Gel Electrophoresis of Labeled Restriction Enzyme Sections
1) Prepare a polyacrylamide gel (20 cmX40 cmX1.5 mm; with four 3-cm wells) according to the following recipe:
Acrylamide (30%; w/v)/Bis(propylacetamide) (0.8%; w/v) 20.3 ml
TBE (10x) 12.5 ml
H2092.2 ml
Mix and filter, then add 750ul 10% (w/v) ammonium persulfate and 60ul TEMED.
2) The gel was polymerized for at least 45 min and then pre-electrophoresed for at least lh at 19 W (~400 V). The gel should not be heated during electrophoresis.
3) Add samples and electrophoresis for the required time. The voltage is slowly increased from 400V to 450-500V.
Note: Xylene cyanide blue migrates with 300-bp restriction fragment on 5% gel.
Elution of DNA from polyacrylamide gels
This method works well on polyacrylamide gels ≤8%.
1) Cut the radioactive strip from the gel plate and minimize the size of the strip.
2) Punch a small hole in the bottom of a l.5-ml plastic microcentrifuge tube with an 18-G gauge needle (pink).
3) Insert the bottom of the perforated microcentrifuge tube into another 1.5-ml microcentrifuge tube (without perforations).
4) Place the cut piece of gel into the previous perforated microcentrifuge tube.
5) Centrifuge the two microcentrifuge tubes together in a microcentrifuge for 15 s. The gelatinous mass should be broken up when it passes through the small hole into the next microcentrifuge tube.
6) Add 300ul of TE and mix by shaking.
7) Incubate overnight at 37°C.
8) Punch a small hole in the bottom of a 1.5-ml plastic microcentrifuge tube with a 20-G gauge needle (yellow). Fill the hole in the bottom of the tube with approximately 200ul (volume after filling) of silica gel. The siliconized glass wool should be packed firmly in the bottom of the tube. The bottom of this tube is then inserted into another 1.5-ml microcentrifuge tube (no wells). Using a 1000-ul pipette tip (blue) with the tip cut off by a razor blade about 2-3 mm, a mixture of TE and crushed pellets is placed on the glass wool, and the device made of the two microcentrifuge tubes is centrifuged in a microcentrifuge for 10 s. The lower tube should contain TE-eluted DNA fragments.
9) Precipitate the probe DNA with ethanol, then dissolve in TE.
10) Store at -20°C.