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Dna Footprinting
DNA footprinting is a technique used to identify where proteins bind to DNA. It was developed in 1978 and works by treating DNA with enzymes or chemicals that cut DNA, except for regions bound by proteins. This leaves a "footprint" where the protein is bound and protects the DNA. There are two main types: DNase I footprinting cuts DNA randomly except where proteins are bound, while DMS footprinting modifies DNA bases except where proteins protect them from modification. The cut or modified DNA is then run on a gel to identify the protein binding site. DNA footprinting is useful for mapping transcription factor binding sites and studying protein-DNA interactions.
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Dna Footprinting
- 1. DNA Footprinting Adarsh P P M.Sc.Bioinformatics Union Christian College, Aluva
- 2. What is DNA footprinting???? Powerfuland fairly rapid methods for mapping where and how proteins bind tightly to DNA
- 3. HISTORY • Developed byDavid Galas and Albert Schmilz in 1978 • Used to study the binding of lac repressor • Modification of Maxam-Gilbert chemical sequencing technique
- 4. PRINCIPLE • Assay usedto identify the site of protein-DNA interaction. • It has been found that a protein bound to the DNA protect it from the action of degradative enzymes. • The main application of this method is to find the binding regions of DNA transcription factors.
- 5. PROCEDURE • The DNAof interest is first amplified using PCR • The double stranded amplified DNA is labelled at one end of each strand. • Next, the protein of interest is added. • The DNA is then cleaved by chemical or enzymatic cleavage agent. • The fragments are then run on a denaturing polyacrylamide gel.
- 7. There are mainlytwo types of DNA footprinting: • DNAse I footprinting • DMS footprinting
- 8. DNAse I Footprinting •Here the DNAse I enzyme is used • It not only helps to find the target protein that binds to specific DNA, but also identify which sequence to which the protein is bound. • The protein binding to the DNA protect it from DNAse I • The fragments are then left after cleavage, so it can be sequenced
- 9. METHOD • Prepare end-labelledDNA • Bind the protein • Mild digestion with DNAse I (randomly cleaves dsDNA on each strand) • Separate DNA fragments on denaturing acrylamide gels
- 10. • Samples inlane 2-4 had increasing concentration of DNA-binding protein.
- 11. DIMETHYLSULFAT E FOOTPRINTING • DMSinduces methylation of guanine residues. • Similar to DNAse footprinting • Mildly treat the DNA with DMS (after the addition of protein) so that on an average only one methylation occurs per DNA. • The DNA is then treated with a reagent that removes the methylated purins. • The apurinic sites thus are removed with the help of cleaving agents. • Each band end next to a nucleotide that was methylated and thus unprotected by the protein.
- 12. METHOD • End-labelled DNAfragment • Bind protein • Treat with DMS, methylates purines • Partially cleave DNA at methylated bases. • Separate fragments on the gel
- 13. • Lane 1and 4 have no protein • Lane 2 and 3 have different amount of protein. Disadvantages • Protein binding protects some purines from modification by DMS, it but can stimulate modification of others (helix distorted or partially melted) • Cannot be used to detect the proteins binding to the AT rich sequences.
- 14. APPLICATIONS • In vivoFoot printing • Quantitative Footprinting • Detection by capillary electrophoresis.
- 15. In vivo Footprinting • Used to analyse protein-DNA interaction occurring at a given time in a cell. • The cell membrane is first permeabilized using UV rays. • Then the DNAse I is used. • After the cleavage, the single stranded DNA is isolated and purified, a linker DNA is added into the break points. • The region is then amplified and run on gel. • This method can be coupled with immunoprecipitation The DNA binding to the protein of interest can be precipitated with an antibody to the protein, and then specific region of protein binding can be assessed.
- 16. Quantitative Footprinting • Modificationof normal footprinting • Varying concentration of protein is used • Protein binding affinity can be estimated, because the formation of footprinting can be observed with increasing concentration of protein
- 17. Detection by Capillary Electrophoresis •Capillary electrophoresis device is used. • The PCR primers are coupled with carboxyflourescein, thus the fragments produced by digestion will contain carboxyflourescein • This fluorescence can be detected using capillary electrophoresis device • Transcription factor binding sites can be effectively identified.
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