Shotgun DNA Mapping<br />Anthony Salvagno<br />
Shotgun DNA Mapping<br />Prove that we can map DNA fragments in genome<br />Unzipped DNA has a unique signature<br />Want ...
DNA: The Code of Life<br />Double stranded flexible polymer<br />Covalently bonded sugar molecules make up the backbone<br...
Why do we care about DNA?<br />DNA controls every aspect of who we are!<br />Proteins bound to DNA can explain a lot about...
Power of Single Molecule Analysis<br />Red Line – protein bound to DNA<br />Black Line – naked DNA<br />Black Dotted Line-...
Optical Trap<br />Bead is tiny dielectric sphere<br />Laser focus creates large E-field gradient<br />Bead attracted to ce...
Comparison of Unzipped DNA to Simulated Unzipping<br />Simulated unzipping of pBR322 plasmid<br />Simulated plasmid data h...
The Unzipping Construct<br />We can use a similar setup for <br />stretching DNA experiments as well…<br />
Latest Results: Stretching DNA<br />3<br />4<br />2<br />Force<br />1<br />Distance<br />dsDNA stretches with minimal effo...
Coming Soon: DNA Unzipping<br /><ul><li>Tether unzipping construct to glass
Attach bead to DNA
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Shotgun DNA Mapping for IGERT

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This is a talk I gave to the IGERT fellows discussing my project in ~15min.

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Shotgun DNA Mapping for IGERT

  1. 1. Shotgun DNA Mapping<br />Anthony Salvagno<br />
  2. 2. Shotgun DNA Mapping<br />Prove that we can map DNA fragments in genome<br />Unzipped DNA has a unique signature<br />Want to match unzipped DNA to library of simulated unzipping curves<br />Library of Simulated Curves<br />Random fragment<br />Experimental Force<br />Endonuclease<br />Genomic DNA<br />Correct Match<br />dsDNA anchor<br />Step 1: Digest genome into fragments<br />Step 2: Unzip fragment and record forces<br />Step 3: Compare experimental forces to a library of simulated curves<br />
  3. 3. DNA: The Code of Life<br />Double stranded flexible polymer<br />Covalently bonded sugar molecules make up the backbone<br />Hydrogen bonded bases join two strands of DNA<br />There are 4 bases<br />Whyfiles.org<br />
  4. 4. Why do we care about DNA?<br />DNA controls every aspect of who we are!<br />Proteins bound to DNA can explain a lot about their function<br />DNA compaction<br />Transcription<br />Mutations<br />Looking at naked DNA can also diagnose potential problems<br />Deletions<br />inversions<br />
  5. 5. Power of Single Molecule Analysis<br />Red Line – protein bound to DNA<br />Black Line – naked DNA<br />Black Dotted Line- predictions of protein locations<br />F<br />F<br />Unzipping can detect proteins bound to DNA<br />Koch et al. 2002<br />
  6. 6. Optical Trap<br />Bead is tiny dielectric sphere<br />Laser focus creates large E-field gradient<br />Bead attracted to center of focus<br />Want High NA for better trapping<br />
  7. 7.
  8. 8. Comparison of Unzipped DNA to Simulated Unzipping<br />Simulated unzipping of pBR322 plasmid<br />Simulated plasmid data hidden inside simulation of entire yeast genome<br />Comparison with actual plasmid unzipping matches only the simulated plasmid data!<br />A<br />Correct Match, Score 0.2<br />18<br />Force (pN)<br />12<br />0<br />1500<br />Unzipping fork index (bp)<br />B<br />Mismatch, Score 0.8<br />18<br />Force (pN)<br />12<br />0<br />1500<br />Unzipping fork index (bp)<br />
  9. 9. The Unzipping Construct<br />We can use a similar setup for <br />stretching DNA experiments as well…<br />
  10. 10. Latest Results: Stretching DNA<br />3<br />4<br />2<br />Force<br />1<br />Distance<br />dsDNA stretches with minimal effort<br />Limits of DNA flexibility reached, hard to stretch, base-pairs break apart<br />dsDNA overstretches – basically stretching two single strands of DNA<br />DNA breaks or bead detaches from DNA<br />
  11. 11. Coming Soon: DNA Unzipping<br /><ul><li>Tether unzipping construct to glass
  12. 12. Attach bead to DNA
  13. 13. Pull with twezers</li></li></ul><li>Thank You Everyone!<br />sley<br />Lab<br />
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