Hello There, DNA Footprinting Is A Molecular Biology Technique With Wide Applications In Many Areas Of Biological Sciences And Importantly It Is Used For Crime Detection In Forensic Sciences. In This Presentation, You Will Learn What It Is, The Technology, Protocol, Pictorial Representation, Applications And References For Further Study.

1. DNA FOOTPRINTING
LOGESWARAN KA
P20BIT1013
I MSC BIOTECHNOLOGY
PERIYAR UNIVERSITY

2. INTRODUCTION
INTERESTING INVITRO MOLECULAR BIOLOGY TECHNIQUE.
FIRST DISCOVERED BY DAVID GALAS AND ALBERT SCHMITZ IN
THE YEAR 1978. INITIALLY, THEY DEVELOPED TO STUDY BINDING
SPECIFICITY OF Lac REPRESSOR PROTEIN.
ALSO CALLED AS “DNase I FOOTPRINTING”.
IT IS USED TO FIND OUT PROTEIN BINDING REGIONS OF DNA
MOLECULE.
IT IS A MODIFICATION OF MAXEM-GILBERT SEQUENCING.

3. PRINCIPLE
NUCLEASES LIKE DNase I IS USED FOR DEGRADATION OF DNA.
PROTEINS WILL BIND TO DNA AND IT WILL PROTECT THEM
FROM DEGRADATION.
DURING AUTORADIOGRAPHY, THE PROTECTED REGION WILL
FORM A GAP (NO BAND) AND IT IS CALLED AS FOOTPRINT.
THUS, THE SEQUENCE CAN BE DETERMINED BY COMPARATIVE
ANALYSIS.
IN THIS MANNER, DNA FOOTPRINTING IS A SPECIFIC, ACCURATE
AND WIDELY USED TECHNIQUE TO STUDY DNA-PROTEIN
INTERACTIONS.

4. MATERIALS
DNA  100-400 BP LONG PCR AMPLIFIED DNA.
DNA LABELING  THE ENDS OF DNA IS LABELED WITH
RADIOACTIVE COMPOUNDS. THE LABELED DNA IS PURIFIED AND
UNBOUND PROBES ARE REMOVED.
PROTEIN  PURE PROTEIN SAMPLE IS USED AND THE
REACTION WILL BE PERFORMED IN PROTEIN BINDING BUFFER.
CONTROLAND TEST  CONTROL (ONLY DNA) AND TEST (DNA
AND PROTEIN) SAMPLE TUBES WERE PREPARED.

5. CLEAVAGE AGENTS  DNase I IS THE CONVENTIONAL AND
WIDELY USED ENDONUCLEASE FOR FOOTPRITING. IT IS A EASY
TO HANDLE ENZYME. IT WILL NOT BIND TO ADJACENT (NEARBY)
DNA-PROTEIN COMPLEX BECAUSE OF PHYSICAL HINDRANCE
CAUSED BY LARGER SIZE.
PAGE SETUP  POLYACRYLAMIDE GEL ELECTROPHORESIS IS A
PROTEIN SEPARATION TECHNIQUE. IT IS USED TO SEPARATE
DIGESTED FRAGMENTS.
AUTORADIOGRAPHY  IT IS PERFORMED TO ANALYSE THE
BANDS AFTER THE COMPLETION OF GEL RUN AND THEN
COMPARATIVE ANALYSIS WILL BE PERFORMED.

6. PROCEDURE
PCR AMPLIFICATION OF DNA SAMPLE.
RADIOACTIVE 5’ END LABELING OF DNA (USING 32P) WHICH HAS
PREDICTED PROTEIN BINDING SITES.
PREPARATION OF CONTROL (DNA ONLY) AND TEST TUBES (DNA
AND PROTEIN).
IMMUNOPRECIPITATION USING SPECIFIC ANTIBODY AGAINST
PROTEIN OF INTEREST AND SEPARATION OF DNA-PROTEIN
COMPLEX FROM THE UNBOUND PROBES.

7. ADDITION OF CLEAVAGE AGENTS (DNase I) TO BOTH CONTROL
AND TEST TUBES AND THEN THE REACTION IS PERFORMED IN
PROTEIN BINDING BUFFER UNDER PROPER CONDITIONS.
PAGE IS PREPARED AND BOTH CONTROL AND TEST SAMPLES
ARE ADDED TO THEIR RESPECTIVE WELLS ON THE GEL AND
THEN IT IS ALLOWED TO RUN.
AFTER THE COMPLETION OF GEL RUN, THE BANDS ARE
OBTAINED BY AUTORADIOGRAPHY AND THEN COMPARATIVE
ANALYSIS OF CONTROL AND TEST SAMPLES IS PERFORMED.

8. RESULT
THE CONTROL SAMPLE WILL FORM A UNIFORM CONTINUED
LADDER OF BANDS IN THE AUTORADIOGRAPH SINCE IT
CONTAINS ONLY THE DNA SAMPLE.
THE TEST SAMPLE WILL FORM A UNEQUAL LADDER OF BANDS
WITH GAPS OR FOOTPRINTS. FOOTPRINT IS THE PROTECTED
REGION FROM DEGRADATION AND IT INDICATES THE GAP
REGION IS A POTENTIAL PROTEIN BINDING SITE.

10. APPLICATIONS
SEQUENCE SPECIFIC PROTEIN BINDING SITES IDENTIFICATION.
DNA-PROTEIN INTERACTIONS STUDY.
TRANSCRIPTIONAL REGULATIONS STUDY.
PROMOTOR, OPERATOR AND SILENCE SEQUENCES OF GENE
IDENTIFICATION.
FUNCTIONAL GENES IDENTIFICATION.
HORMONE RESPONSE ELEMENTS IDENTIFICATION.

11. REFERENCES
1. https://academic.oup.com/nar/article-
abstract/5/9/3157/2380868?redirectedFrom=fulltext
2. https://bitesizebio.com/38946/dna-footprinting/
3. https://www.creativebiomart.net/resource/principle-protocol-dnase-i-
footprinting-377.htm
4. https://www.biologyexams4u.com/2014/02/dna-footprinting-
definitionprinciple.html

12. THANKS FOR WATCHING