1.
Seminary presented by: Santiago Álvarez Rivera
Medicine - Molecular Biology - 3rd Semester - UPB

2.
INTRODUCTION
MICROBIAL TRANSCRIPTION FACTORS
They are proteins capable of specifically binding to
short DNA sequences located in gene promoters, and
these which activate or repress transcription of target
genes typically in response to an environmental or
cellular trigger.
They work as regulators of transcription
Photo taken from: https://www.news-medical.net/life-
sciences/Role-of-Transcription-Factors.aspx

3.
INTRODUCTION
RESTRICTION ENZYMES
A restriction enzyme, also called restriction
endonuclease, is an endonuclease that can recognize
a characteristic sequence of nucleotides within a DNA
molecule and cut the DNA at that specific point.
Different restriction enzymes recognise and cut
different DNA sequences.
Photo taken from:
https://www.nagwa.com/en/videos/516142168739/

4.
GENERAL
OBJECTIVE
EXPOSE THE USE OF RESTRICTION ENDONUCLEASE,
PROTECTION, SELECTION AND AMPLIFICATION TO
IDENTIFY PREFERRED DNA-BINDING SEQUENCES OF
MICROBIAL TRANSCRIPTION FACTORS

5.
METHODS
REPSA: Restriction Endonuclease, Protection,
Selection, and Amplification. REPSA consists of
several “rounds” in which a set of DNAs is incubated
with a transcription factor of interest and then
challenged with an IISRE (which is type IIS restriction
enzymes)
DNA PURIFICATION:
Depending on your tissue or cell samples, you can use
chemical treatment, enzymatic digestion or
mechanical disruption to lysate the DNA, and remove
contaminants from the nucleic acids. This have the
purpose of make it convenient for storage and
handling for the investigation.
Photo taken from:
https://science.howstuffworks.com/life/cellular-
microscopic/dna.htm

6.
METHODS PCR: is a laboratory technique for rapidly
producing (amplifying) millions to billions of
copies of a specific segment of DNA, which can
then be studied in greater detail.
PCR enables the synthesis of specific DNA
fragments using a DNA-polymerase enzyme,
which takes part in the replication of the cellular
genetic material.
RESTRICTION ENZYMES:
Type IIS restriction enzyme.
Recognize a specific sequence of DNA and cut it
there.
Photo taken from:
https://labmal.com/2019/08/05/restriction-enzyme/

7.
RESULTS

8.
RESULTS

9.
DISCUSSION
AUTHOR AUTHOR WORDS AGREE OR
DISAGREE
Wah D.A. “It is worth noting that the mechanism of action for
FokI cleavage requires two FokI monomers to
interact at the cleavage site, and this preferentially
occurs between DNA-bound monomers.”
Hardenbol P “Although this protocol describes the use of REPSA
for transcription factor DNA-binding specifically,
REPSA can be used with a variety of other ligands,
including small molecules and nucleic acid”
Vashisht Gopal YN Notably, REPSA is not exclusive to transcription
factor ligands and has been experimentally validated
to find DNA-binding sequences for nontranscription
factor proteins, nucleic acids, and small molecules

10.
CONCLUSIONS 1. REPSA technique is in fact a functional
technique to identify those DNA binding
sequences, and with them we can continue
investigating and deepening about the
transcription regulatory network, based in it
biological function.
2. In medicine, this is very important
because many diseases have a relation with
dysregulation of transcription factors, like
cancer, so with this technique, we can be
more precise with those diseases and also
with therapeutic solutions for them.
Photo taken from: https://lab-
biomolecular.com/2019/09/03/la-monitorizacion-del-
cancer-mediante-el-analisis-de-adn-circulante-podria-
optimizar-los-tratamientos/