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Lecture 17

BIOL 5060 Lecture Notes - Lecture 17: California Institute Of Technology, Dna-Binding Domain, Phosphodiester Bond

6 pages50 viewsSpring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
17

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3/23_RecombinantDNATechnology_Hoffman3/23/2017 2:00:00 PM
Protein-DNA interactions
DNaseI footprinting- will show region of protein binding site
o GGATCC- palindrome; BAHI site
o Test the sequence specificity of the interaction by making cold
competing DNA molecules
o Do a band shift…
1. Probe + protein
2. Probe + protein + 5x cold probe
1/5th the intensity
Detects the radioactive
o Order oligos that are identical to the wild-type restriction site
with flanking sequences with a slight mutation along the
sequence
Use these mutated strands as the cold competing DNA
What if cold species have no effect?
What would the PCR result look like if they did have an
effect?
Is a particular protein in the protein-DNA complex?
o Super-shift experiment- two approaches to this
1. Translational fusions- Carry out EMSA with an extract
making wild-type protein versus an extract making a
hybrid version of the protein
Translational fusion produce hybrid/fusion
proteins
If it’s a functional fusion protein, it should shift
Would prove that protein is part of the
complex because fusion protein affected
shifting
If the fusion protein loses binding activity no
band
No shift would tell us that fusion protein
affected binding activity PROTEIN IS A
PART OF THE COMPLEX
If fusion or wild-type protein isn’t involved in
binding, there will be no shift
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Protein is not part of the complex (bands
are unaffected by fusion and look them
same)
2. Super-shift using antibodies- you can add a large
amount of proteins to the complex
Carry out binding reaction using extract
containing tagged protein plus and minus
antibody to tag
If antibody binds protein of interest…
Super shift will either shift higher protein
is in the protein-DNA complex
Band will be eliminated protein is part of
the complex and the binding of the antibody
didn’t allow binding
Now we want to know…
What protein binds our DNA? What sequence(s) can my protein
bind?
Use SELEX- create a population of DNA molecules and screen it for
sequences our protein binds to
o Create a library of random DNA sequences with PCR
o Forward (F) oligo
o Long oligo- has the F sequence, 26 random nucleotides,
complement of R
o Reverse (R) oligo
PCR with these 3 oligos like a library screen
o N26 sequence= the insert
o
library of sequences
Bind protein of interest to a column or use soluble protein
o
carry out a binding reaction
o if you do a column you can then wash it and dilute the DNA
o if its done in a soluble reaction you can pass the binding
reaction over a filter elute the DNA from that
o **Generally, let the protein bind your DNA and then separate
out/// elute DNA of interest and REPEAT (7 or so cycles of
enrichment)
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