The technique used to identify regions where proteins are bound to DNA

1. DNA
Footprinting
Adarsh P P
M.Sc. Bioinformatics
Union Christian College, Aluva

2. What is DNA
footprinting????
Powerful and fairly rapid methods for
mapping where and how proteins bind tightly
to DNA

3. HISTORY
• Developed by David Galas and
Albert Schmilz in 1978
• Used to study the binding of lac
repressor
• Modification of Maxam-Gilbert
chemical sequencing technique

4. PRINCIPLE
• Assay used to 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 DNA of 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 mainly two 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-labelled DNA
• Bind the protein
• Mild digestion with DNAse I
(randomly cleaves dsDNA
on each strand)
• Separate DNA fragments
on denaturing acrylamide
gels

10. • Samples in lane 2-4 had
increasing concentration of
DNA-binding protein.

11. DIMETHYLSULFAT
E FOOTPRINTING
• DMS induces 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 DNA fragment
• Bind protein
• Treat with DMS, methylates
purines
• Partially cleave DNA at
methylated bases.
• Separate fragments on the
gel

13. • Lane 1 and 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 vivo Foot printing
• Quantitative Footprinting
• Detection by capillary
electrophoresis.

15. In vivo Foot printing
• 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
• Modification of 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.

18. THANK YOU