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DNase Foot Printing: Identification of Protein Binding Site of DNABY: Muniba Safdar | Category: Genetics | Submitted: 2010-07-27 06:21:15
Article Summary: "What is DNA foot printing? It is a technique used to identify protein binding site of DNA. As DNA exists in a cell in the form of nucleoprotein complex. Proteins interact with the DNA for the protection of DNA. It means there is DNA-protein interaction in the cell. There are specific sites where proteins bind..."
DNA foot printing is a technique used to identify protein binding site of DNA. As DNA exists in a cell in the form of nucleoprotein complex. Proteins interact with the DNA for the protection of DNA. It means there is DNA-protein interaction in the cell. There are specific sites where proteins bind. Now, we have to identify that site.
Step 1: DNA strand
• Our source is recombinant DNA/cloned DNA.
• Usually, we consider 100-300 base pairs.
• We will take different fragments starting with same point and ends with the same pint.
• After getting a strand of DNA, we will label only one strand.
• Labeling of DNA will be at 5´ end.
• Label with phosphorous 32 (P32) and phosphorous 35 (P35).
• P35 gives glow to DNA.
Step3: DNase I
• DNase I, an enzyme, which only cuts one strand from 5´ to 3´ end.
• Best cleavage agent.
• After labeling of DNA, they are then added with DNase I.
• It randomly cuts the strands.
• Concentration of DNase I should be low so that only one cut in one molecule.
Step4: Run on gel
• Now, cleaved DNA in the absence of DNA binding protein is compared to the cleaved DNA in the presence of a DNA binding protein.
• That site will be protected from enzymatic cleavage where protein bound to the DNA.
• Protection from enzymatic cleavage would result in a fine clear are on the gel which is referred to as "foot print of a protein".
• Through this technique we come to know which sequence is present here.
If we do some modifications in the above steps we will get a technique called EMSA, Electrophoretic Mobility Shift Assay. In this technique
• Particular length of DNA is used.
• Restriction fragments.
• Mild concentration of DNase I.
• Gel used is of good/high resolution that can differentiate small distances.
• We can use urea in a gel to denature.
• Only labeled fragment will glow under radiography.
• Protein has to give more time when bind with DNA.
Why we prefer this technique?
• If we do not have DNase I we can use chemicals, guanosine or any other reagent.
• If there is a site present between two base pairs it can determine that site.
Why we do not prefer this technique?
• It is very tedious (time consuming).
• Technically demanding. For example, pH changes, cut site, etc).
• We are basically using DNases and its cofactor is Mg++ and we must have to use it, because without this cofactor it will not work.
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