DNA Footprinting

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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. CONTROL AND 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 32 P) 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 .

9. 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.

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

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