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Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
Thorough Intro To SDM For Osley Lab (82109)
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Thorough Intro To SDM For Osley Lab (82109)

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I made this presentation to show some collaborators whose lab I worked in last semester. In it I discuss the past, present, and future of Shotgun DNA Mapping and all that it contains.

I made this presentation to show some collaborators whose lab I worked in last semester. In it I discuss the past, present, and future of Shotgun DNA Mapping and all that it contains.

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Transcript

  • 1. A Thorough Introduction to Shotgun DNA Mapping and Kicking Ass in Science
    I made this…
    Presents:
    By Anthony
    …and this
  • 2. Thank You Osley Lab
    A big thanks to Kelly and Mary Ann…
    Thanks Cory and Toyoko…
    …and KochLab
  • 3. 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 histoneoctamer, the optical tweezer allows for counting the remaining nucleosomes at the end of the remodeling process.”
    Reassembled Nucleosomes
    RNA Pol II
    promoter
    cryptic
    promoter
    Transcription
  • 4. Shotgun DNA Mapping in a Nutshell
    What this talk is mostly about
    Austin is in there too
    Procedure
    Library of Simulated Curves
    Random fragment
    Experimental Force
    Endonuclease
    Genomic DNA
    Correct Match
    dsDNA anchor
    Step 1: Digest genome into fragments
    Step 2: Unzip fragment and record forces
    Step 3: Compare experimental forces to a library of simulated curves
  • 5. 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.
  • 6. How Matching Works
    • Using known physical models you can predict (simulate) what the force profile of unzipped dsDNA would look like
    • 7. You then can compare that to actual unzipping data
    • 8. Correct matches yield match scores close to zero, just how Larry defined it
    A
    B
    Correct Match, Score 0.2
    Mismatch, Score 0.8
    18
    18
    Force (pN)
    Force (pN)
    12
    12
    0
    1500
    0
    1500
    Unzipping fork index (bp)
    Unzipping fork index (bp)
  • 9. 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!
  • 10. How do Tweezers Work?
    We can measure forces on the bead based on deflections
    observed by a QPD (quadrant photodiode).
  • 11. Our Optical Tweezers
    movie of tweezer setup
  • 12. 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
  • 13. Yeast Cell
    Spheroplasting
    RNaseA-ing
    Phenol/Chloroform Extraction and Ethanol Precipitation
    It’s foreign so it’s gotta be evil
  • 14. Next Step
    Need digested plasmid DNA and digested genomic DNA
    Want to clone fragments
    For sequencing
    So we can unzip a lot of fragments
    Michael Bay’s next film… too late I already sold the rights
  • 15. The first of many gels
    Lanes:
    1: pRS413 uncut
    2: pRS cut with XhoI
    3: pRS cut with NotI
    4: pRS cut with BstXI
    5: genomic uncut DNA (gDNA)
    6: gDNA cut with XhoI
    (didn’t work)
    10kb length
    My archnemesis
  • 16. Digested gDNA
    Lanes:
    1: Uncut gDNA
    2: gDNA cut with XhoI
    3: gDNA cut with XhoI (for redundancy)
    Get used to this, there is a lot more coming
    Making this was really annoying
  • 17. Inserting DNA
    CIP – Calf Intestinal Phosphatase
    T4 DNA Ligase - ??? DNA Ligase
    Terminators can’t self terminate.
  • 18. 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
  • 19. Transformation Success?
    E. Coli DNA
    Extracted plasmid DNA
    This is all Koch’s fault
  • 20. Double Digest and pBluescript
    I was drunk when I took this picture
    I was drunk when I slept with this one
  • 21. Redoing with pBS
    Now that is definitely some random genomic fragments
    Top Image quick extraction
    Bottom Image is good extraction
    I like pink tape
  • 22. 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
    Not for Larry’s Eyes
    I thought it would be funny if I used a print screen of this slide for this slide.
  • 23. 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
    BstXI
    pRL fragment
    NotI
    NotI hairpin
    or
    SapI
    Top and bottom
    Annealed oligos
    NotI end
    SapI end
    The sequel to Michael Bay’s movie
    Rights also already sold
  • 24. When it’s all done
    More on next slide
    gDNA
    plasmid
    Biotinylated fragment
    Digitylated fragment

    Gel of Digested Fragments
    This is what skittles does to your DNA
    The quality of this image is a direct result of a computer from 1991
  • 25. What I have now
    What it should look like
    What it looks like
    both
    fragment
    anchor
    1991 strikes again
    2009 artist rendition
  • 26. Combine with Fragments
    Ligate the plasmid random fragments to the tethering construct
    Use flow chamber fluidics to prepare sample for tweezing
    Tweeze
  • 27. The Future
    Unzipping Nucleosomes
    Unzipping RNA Pol II from two directions
    Hope is that it will provide different unzipping signatures
  • 28. No Mas

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