dna footprinting is a method of investigating the sequence specific of dna binding protein in vitro.

1. DNA FOOTPRINTING

2. INTRODUCTION
• DNA footprinting is a method of investigating the sequence specific of
DNA – binding protein in vitro.
• This technique can be used to study protein – DNA interactions both
outside and within cell.
• Techniques like DNA footprinting help elucidate which protein bind to
these associated region of DNA and unravel the complexities of
transcriptional control.

3. HISTORY
• In 1978,David galas and Albert Schmitz developed the DNA
footprinting technique to study the binding specificity of the lac
repressor protein .
• It was originally a modification of the 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 foot print.

5. METHOD
The simplest application of this technique is to assess whether a given
protein binds to a region of interest within a DNA molecule
• Polymerase chain reaction(PCR) amplify and label region if interest
that contain a potential protein- binding site.
• Add protein of interest to a portion of the labelled template DNA ; a
portion should remain separate without protein ,for later comparison
• Now, add a cleavage agent to both the portion of DNA template
(cleavage agent is a chemical or enzyme that will cut at random
locations in a sequence independent manner).

6. • Run both the samples side by side on a polyacrylamide gel
electrophoresis .
• The portion of DNA template without protein bill will be cut at
random locations , and thus when it is run on a gel ,will produce a
ladder- like distributions.
• The DNA template with a protein will result in ladder distribution with
a break in it , the “footprint” , where the DNA has been protected
from the cleavage agent .

7. Labeling
• The DNA template can be labeled at the 3’ or 5’ end , depending on
the location of binding sites .
• Labels that can be used are : radioactivity and fluorescence .
• Radioactivity has been traditionally used to label DNA fragments for
footprinting analysis.
• Radioactive labelling is very sensitive and is optimal for visualising
small amount of DNA .
• Fluorescence is a desirable advancement due to the hazards of using
radio-chemicals .

8. • However, it has been difficult to optimize because it is not always
sensitive enough to detect the low concentrations of the target DNA
strands used in a DNA footprintings experiments.
• Electrophoretic sequencing gels or capillary electrophoresis have
been successfully in analysing foot printing of fluorescent tagged
fragments .

9. Cleavage agent
• A variety of cleavage agent can be chosen .
• Ideally a desirable agent is one that is sequence neutral , easy to use ,
and is easy to control .
• The following cleavage agent are described in detail : DNase I is a
large protein that function as a double – strand endonuclease .
• It binds the minor group of DNA and cleaves the phospodiester
backbone .it is good cleavage agent for footprinting because its size
makes it easily physically hindered .thus is more likely to have to its
action blocked by a bound protein on a DNA sequence In addition ,
the DNase I enzyme is easily controlled by adding EDTA to stop the
reaction .

10. • Hydroxyl radicals are created from the Fenton reaction , which
involves reducing Fe2+ with H2o2 to form free hydroxyl molecules
.These hydroxyl molecules react with the DNA backbone resulting in a
break. Due to their small size , the resulting DNA footprint has a high
resolution .
• Ultraviolet irradiation can be used to excite nucleic acid and create
photoreactions , which result in damaged bases in a DNA strand.
Advantages of uv are that it reacts with very quickly and therefore
capture the interactions that are only momentary .

11. Applications
• DNA footprinting is often used to identify the binding sites of
proteins in a DNA molecule. Researchers often use this technique to
identify whether a particular protein can activate or inhibit
transcription. In addition, scientists also use this method to detect
where proteins bind to DNA in a living cell.