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Background on DNA Footprinting
DNase footprinting is a molecular biology method that enables the detection of DNA-protein interactions by exploiting the property that a protein interacting with DNA will protect the DNA at that interaction site from restriction digestion or DNAase enzymatic cleavage. A DNA restriction fragment which contains the specific binding site is labeled at one end, usually with 32P and used for footprinting.
Protein and DNA are allowed to hybridize and bind together.
DNA footprinting utilizes the enzyme DNase I or deoxyribonucleic acid nuclease I in order to digest or cut away free radiolabeled (or labelled) DNA leaving the protein bound DNA protected. The DNA restriction fragments are treated lightly with DNase I, which makes single-strand breaks (nicks) in the DNA. A small amount of enzyme is used such that there is an average of less than 1 nick/strand. The reaction is then stopped, the DNA is denatured, and the mixture is run on a denaturing polyacrylamide sequencing gel. These fragments separated by gel electrophoresis are exposed to detect the protected DNA area by analyzing the banding cleavage pattern on the gel.
The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint".
By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed.
DNAse Footprinting Buffer Recipes
DNase Footprinting Binding Buffer Recipe
2X w/o KCl / For 10mls:
Tris-HCl pH7.6: 500 ul of 1M: 50 mM
MgCl2: 125ul of 1M: 12.5mM
EDTA: 2 ul of 0.5M: 1mM
Glycerol: 2 mls: 20%
DTT : 10 ul of 1M: 1 mM
Binding Buffer (Gel Shift buffer)
5X w/o KCL
50mM: Tris-HCL, pH 7.5
0.25mg/ml: poly (dI-dC) poly (dI-dC)
CaCl2: 50 ul of 1M: 5 mM
MgCl2: 100 ul of 1M: 10 mM
0.1 M: NaOH:formamid (1:2 v/v)
0.1%: xylene cyanol
0.1%: bromophenol blue
NaCl: 400 ul of 5M: 200 mM
EDTA: 600 ul of 0.5M: 30 mM
SDS: 1 ml 10%: 1%
TEBe (Tris-EDTA-Beta mercap.)
Tris-HCl, pH 7.6: 500 ul of 1 M: 50 mM
EDTA: 2ul of 0.5 M: 1 mM
Beta-mercaptoethanol: 10 ul : 15mM
10 x K buffer
for 1 ml
Tris-HCl pH 7.5: 100 ul of 1 M: 100 mM
MgCl2: 100 ul of 1 M: 100mM
DTT: 50 ul of 1M: 50 mM
dH2O: 750 ul
DNAse Footprinting Protocol
The DNA used usually contains the binding site of your protein of interest. The DNA is purified, then digested with restriction fragments to be of an appopriate size. The DNA fragment is labeled on one end (binding site > 25 bp from end).
One strand labeling can be accomplised by:
- isolating the fragment with restriction enzyme(s) containing 5' overhang, labeling with Klenow and the appropriate hot nucleotide. Then digest with an enzyme that removes one end.
- isolating a fragment digested with a restriction enzyme that creates 5' and 3' end, labeling with Klenow will only label the 5' overhang.
- As in "a" but with any enzyme and using polynucleotide kinase to label 3' end (kinase) and digesting off one of the ends with a second (third) restriction enzyme.
(REMINDER: if labeling with Klenow, at the end of reaction add an excess of cold nucleotide of the same nucleotide that is labeled ( i.e. if you use dCTP32, add dCTP at the end) to make sure all ends are filled in equally.
For Klenow fragments, 0.3ug bring up to 100 ul in TE, heat kill Klenow at 68 °C for 10 minutes.
LABELING with one 5' overhang
Sample RXN : 15 ng is need for each reaction. If making probe for many reactions JUST increase amount of DNA up to 300 ngs.
15ng: DNA fragment
2 ul: 10x K buffer
5 ul: 32P dCTP 3.33 uM 10 uCi/ul
2 ul: 2mM @ dA, dG dTTP
1 ul: Klenow
20 ul Final volume, 37°C 30 min.
--add 1 ul 10mM dNTP, 5 min @ 37°C .
--add 80 ul TE. 68°C 15 minutes, put on ice.
--G-50 Spin Column (spin at about 2000g)
--ADD 1/10 vol 3M Na0Ac, 2.5 volumes EtOH, Ice 30 mins, (Optional if [DNA] is = or > 3 ng/ul you might post G-50 directly) Microfuge 30 minutes.
--wash with 70%
--Dry Bring up in 5ul
Binding Reaction For DNase Footprinting
Binding Reaction should have between 10 and 150mM KCl (more salt reduces binding but increases specificity). The typical concentration is 50mM.
Protein DNA Binding:
25 ul of 2X Binding Buffer w/o KCl
5ul of 3 ng/ul unilabeled DNA
X ul of KCl to equal 10-150 mM KCl
dH20 to equal 50 ul minus volume for binding protien
1-3 Footprinting Units of DNA binding protein (or 10 to 160 ug of Crude nuclear extract)
--mix gently, ice 10 minutes.
KEEP TIMING IN MIND,
--ADD 50 ul of RT Ca/Mg solution, 18°C for 1 minute.
--ADD 3 ul dilute RQ1 DNase (0.05 u/ul diluted in Tris) INCUBATE 1 minute.
--STOP addition of 90 with 37°C STOP solutions,
--you should Phenol: CIA, and CIA extract, and Ethanol precipitate.
--RESUSPEND in 4ul of Loading Solution.
--Run on 5% standard Urea-DNA sequencing gel (consider wedge gel) with appopriate loading lanes such as DNA ladder, uncut DNA, and controls.
Analyze footprinting pattern.
Troubleshooting and Problems? See the DNAse Footprinting Forum!
For problems and troubleshooting DNAase footprinting, post a new thread to discuss it in the DNA Footprinting Forum!
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