DNA footprinting

DNA footprinting- easy and short summary

1. DNA  footprinting Ms Saajida Sultaana Mahusook

2. DNA  footprinting  is an in-vitro molecular technique used to identify protein binding regions on a DNA molecule . This technique is mainly used to identify the transcription factors that bind to promoter, enhancer or silencer region of gene to regulate its expression. The regulation of transcript ion of a gene can be studied using this method . The technique is also called as DNAse I  footprinting . Thousands of proteins (enzymes) are interacting with DNA in the nucleus for regulating activities like replication, transcription, translation etc.

3. Transcription is a process where the DNA is converted into RNA in a cell nucleus. Initiation of transcript 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. History   In 1978, David Galas and Albert Schmitz developed this  technique to study the binding specificity of the lac repressor protein. It was originally a modification of Maxam Gilbert chemical sequencing technique.

4. Principle: In this technique, nucleases like DNAse I is used which will degrade DNA molecule. Nucleases cannot degrade DNA if it is bounded by a protein. Thus that region is protected from degradation by nucleases. This protected DNA region is called the  footprint .

5. Procedure :

6. Procedure: 1.  DNA fragment (choice of interest) containing 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.

7. 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. (Comparison of both samples reveals foot prints or protein binding sites).

8. Cleavage agents used in DNA  footprinting   are DNase I: DNae I is a double strand endonuclease enzyme that cleaves the phosphodiester bond present in the DNA. The enzyme action can be controlled by EDTA solution. The enzyme acts on  DNA structure and sequence specific, resulting in an uneven ladder. This can also affect the precision of predicting protein's specific DNA sequence . 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 . Ultraviolet radiation: Ultraviolet radiations are used to excite the nucleic acid and this may lead to damaged  DNA  fragments.

9. Application DNA   footprinting  can be used to determine the sequence specific DNA-binding protein site. To study DNA-protein interaction Transcriptional regulations can be studied. To find out regions in a gene where transcription factors (proteins) binds (that is control elements like operators, promoters etc ) and initiates transcriptions. Promoter , enhancer and silencer sequence of a gene can be identified. To identify the functional genes present in the large genome of human . To find out Hormone response elements (HREs) that is specific sequences where hormone receptor complexes bind.

10. Reference http:// www.biologyexams4u.com/2014/02/dna-footprinting-definitionprinciple.html?m=1 http://www.biotecharticles.com/Genetics-Article/DNA-Footprinting-and-Gene-Sequencing-74.html

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