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DNA Footprinting and Gene SequencingBY: Nidhi Uppangala | Category: Genetics | Submitted: 2010-06-02 21:23:32
Article Summary: "DNA footprinting is a molecular technique used to identify the specific DNA sequence (binding site) that binds to a protein. This technique mainly used to identify the tran script ion factors which bind to promoter, enhancer or silencer region of gene to regulate its expression..."
DNA footprinting is a molecular technique used to identify the specific DNA sequence (binding site) that binds to a protein. This technique mainly used to identify the tran script ion factors which bind to promoter, enhancer or silencer region of gene to regulate its expression. Therefore the regulation of tran script ion of a gene can be studied using this method.
Tran script ion is a process where the DNA is converted into RNA in a cell nucleus. Initiation of tran script ion takes place when the enzyme RNA polymerase binds to a gene sequence known as promoter sequence. DNA footprinting can be used to identify RNA polymerase interacting DNA sequence.
DNA footprinting Procedure:
1. DNA fragment thought to contain protein binding sequence is extracted, amplified and labelled at one end of the double helix using polymerase chain reaction technique.
2. Labelled DNA fragments with DNA binding protein and cleavage agent are mixed in a test tube.
3. In another test tube labelled DNA fragments are mixed with cleavage agents without DNA binding protein. This is used as standard to compare the results.
4. Cleavage agent cuts the DNA fragment present in the both test tubes but no cuts are made at the specific region of DNA where proteins are bound. Protein has protected the DNA binding site from cleavage agent.
5. DNA fragments are separated by polyacrylamide gel electrophoresis and visualized using autoradiogram.
6. When compared with the standard missing band (footprint) indicates the protein binding specific DNA sequence.
Cleavage agents used in DNA footprinting are
1. DNase I:
DNae I is a double strand endonuclease enzyme. DNase I cleaves the phosphodiester bond present in the DNA. The enzyme action can be controlled by EDTA solution. The action of the enzyme is DNA structure and sequence specific, resulting in an uneven ladder. This can also affect the precision of predicting protein's specific DNA sequence.
2. Hydroxyl radicals:
Hydroxyl radicals are produced from a reaction where iron salt is reduced with hydrogen peroxide to form free hydroxyl molecule. The free hydroxyl molecule cleaves the DNA fragment. Hydroxyl radicals are independent of DNA sequence for their action, therefore form evenly distributed ladder. But the reaction rates of these radicals are very slow therefore it requires more time to cleave the DNA fragments.
3. Ultraviolet radiation:
Ultraviolet radiations are used to excite the nucleic acid and this may lead to damaged DNA fragments.
Application of DNA Footprinting:
1. DNA footprinting can be used to determine the sequence specific DNA-binding protein site.
2. Interaction between protein and DNA can be studied using this technique both in-vivo and ex-vivo of a cell.
3. Tran script ional regulations can be studied using DNA footprinting technique.
4. Promoter, enhancer and silencer sequence of a gene can be identified
5. Scientists and researchers can use this technique to identify the functional genes present in the large genome of human.
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