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Lecture 17

BIOL 5060 Lecture 17: 3:23_RecombinantDNATech_hoffman

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Department
Biology
Course Code
BIOL 5060
Professor
Hoffman

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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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Description
3/23_RecombinantDNATechnology_Hoffman 3/23/2017 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  1/5 ththe 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 • 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) •  after final elution of DNA of interest…. We can either clone individual molecules (Topo cloning vector) OR next generation sequencing (to sequence a bunch of molecules in a single collection so we don’t need to purify—we can get individual sequence reads out of the collection) o  both techniques will give us a population of sequences • Now we can do bioinformatics to look for enriched sequences What protein binds our sequence? (no candidate protein) • Could depend on the abundance of the protein o if its highly abundant you can purify the binding activity ▪ Use EMSA as the assay to know what fractions your protein is in ▪ Purified protein sequence determined by mass spectrometry o Low abundance ▪ Detection of in vitro binding activity in a library screen  cDNA library in phage vector- do a plaque lift where the detection is labeled DNA to which the protein binds • look for binding activity in a plaque • NOT HYBRI
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