Thorough Intro To SDM For Osley Lab (82109)
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A Thorough Introduction to Shotgun DNA Mapping and Kicking Ass in Science
By Anthony
Presents:
I made this…
…and this
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Thank You Osley Lab
• A big thanks to Kelly and Mary Ann… • Thanks Cory and Toyoko…
…and KochLab
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Motivation• Need better ways to study native chromatin• Single-molecule analysis can have a huge
impact• Kornberg said so:
“By pulling at the DNA with forces strong enough to unwrap DNA from the histone octamer, the optical tweezer allows for counting the remaining nucleosomes at the end of the remodeling process.”
RNA Pol II
Transcription
Reassembled Nucleosomes
promoter
crypticpromoter
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Shotgun DNA Mapping in a NutshellProcedure
Step 1: Digest genome into fragments
Step 2: Unzip fragment and record forces
Step 3: Compare experimental forces to a library of simulated curves
Genomic DNA
Endonuclease
dsDNA anchor
Random fragment
Experimental Force
Library of Simulated Curves
Correct Match
Austin is in there too
What this talk is mostly about
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How will it work on native chromatin?
• Unzip with everything attached
• Allow dsDNA to rezip by relaxing strand
• Unzip naked DNA• Use matching program
to locate strand and binding sites in genome
dsDNA Bound protein etc.
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How Matching Works
12
18
Forc
e (p
N)
0 1500Unzipping fork index (bp)
Simulated data
Optical tweezersData (Koch 2002)
SimulationOT Data
Correct Match, Score 0.2
0 1500Unzipping fork index (bp)
Simulated data
Optical tweezersData (Koch 2002)
OT DataSimulation
Mismatch, Score 0.8
12
18Fo
rce
(pN
)A B
•Using known physical models you can predict (simulate) what the force profile of unzipped dsDNA would look like•You then can compare that to actual unzipping data•Correct matches yield match scores close to zero, just how Larry defined it
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More about matching
• When actual and simulated unzipping events are compared we can see one distinct match
• Test of 32 tethers of same sequence of unzipped DNA worked every time!
File Number (Arb.)
Mat
ch Sc
ore
Match
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How do Tweezers Work?
We can measure forces on the bead based on deflectionsobserved by a QPD (quadrant photodiode).
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Our Optical Tweezers
movie of tweezer setup
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Where do you start?
• Need genomic DNA from yeast
• Grow some yeast• Extract the DNA• Now we’re Koching
A blurry image of yeast cells
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Yeast Cell
• Spheroplasting• RNaseA-ing• Phenol/Chloroform
Extraction and Ethanol Precipitation
It’s foreign so it’s gotta be evil
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Next Step
• Need digested plasmid DNA and digested genomic DNA
• Want to clone fragments– For sequencing– So we can unzip a lot of
fragmentsMichael Bay’s next film… too late I already sold the rights
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The first of many gels
• Lanes:1: pRS413 uncut2: pRS cut with XhoI3: pRS cut with NotI4: pRS cut with BstXI5: genomic uncut DNA
(gDNA)6: gDNA cut with XhoI (didn’t work)
10kb length
My archnemesis
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Digested gDNA
• Lanes:1: Uncut gDNA2: gDNA cut with XhoI3: gDNA cut with XhoI (for redundancy)
Making this was really annoying
Get used to this, there is a lot more coming
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Inserting DNA
• CIP – Calf Intestinal Phosphatase
• T4 DNA Ligase - ??? DNA Ligase
Terminators can’t self terminate.
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Making Clones
• Mix Competent E. coli cells with plasmid DNA
• E. coli readily replicates plasmid
• Grow cells on petri dish• Cells grow into
individual colonies• If plasmid has inserts
then each colony is a separate insert
One of them likes pizza
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Transformation Success?
This is all Koch’s fault
E. Coli DNA
Extracted plasmid DNA
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Double Digest and pBluescript
I was drunk when I took this pictureI was drunk when I slept with this one
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Redoing with pBS
• Now that is definitely some random genomic fragments
• Top Image quick extraction
• Bottom Image is good extraction
I like pink tape
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Sequencing
• Involves some steps I don’t know
• Need to sequence so that when we unzip we can know what the correct match is
• Larry look away
I thought it would be funny if I used a print screen of this slide for this slide.
Not for Larry
’s Eyes
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Tether Construction
• Make an oligo that has BstXI site and is Biotinylated
• We made 2:– One is a hairpin with a NotI
site– The other is two single
stranded oligos with a SapI site
• Remember our fragments have both NotI ends and SapI ends
pRL fragmentBstXI
or
NotI
SapI
NotI hairpin
Top and bottomAnnealed oligos
NotI end
SapI end
The sequel to Michael Bay’s movieRights also already sold
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When it’s all done
• More on next slide
…
gDNA plasmidBiotinylated fragmentDigitylated fragment
This is what skittles does to your DNAGel of Digested Fragments
The quality of this image is a direct result of a computer from 1991
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What I have now
1991 strikes again
anchor
fragment
both
What it should look likeWhat it looks like
2009 artist rendition
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Combine with Fragments
• Ligate the plasmid random fragments to the tethering construct
• Use flow chamber fluidics to prepare sample for tweezing
• Tweeze
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The Future
• Unzipping Nucleosomes• Unzipping RNA Pol II from two directions
– Hope is that it will provide different unzipping signatures
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No Mas