Shotgun DNA mapping involves three steps: 1) digesting genomic DNA into fragments, 2) unzipping the fragments and recording the force curves, and 3) comparing the experimental force curves to a library of simulated curves to match fragments to locations in the genome. The document discusses how unzipping DNA can detect proteins bound to it based on deviations in the force curve from naked DNA. It presents results matching an experimental plasmid unzipping force curve only to the corresponding simulated plasmid curve within a library simulating an entire yeast genome.

1. Shotgun DNA Mapping Anthony Salvagno

2. Shotgun DNA Mapping Prove that we can map DNA fragments in genome Unzipped DNA has a unique signature Want to match unzipped DNA to library of simulated unzipping curves 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

3. DNA: The Code of Life Double stranded flexible polymer Covalently bonded sugar molecules make up the backbone Hydrogen bonded bases join two strands of DNA There are 4 bases Whyfiles.org

4. Why do we care about DNA? DNA controls every aspect of who we are! Proteins bound to DNA can explain a lot about their function DNA compaction Transcription Mutations Looking at naked DNA can also diagnose potential problems Deletions inversions

5. Power of Single Molecule Analysis Red Line – protein bound to DNA Black Line – naked DNA Black Dotted Line- predictions of protein locations F F Unzipping can detect proteins bound to DNA Koch et al. 2002

6. Optical Trap Bead is tiny dielectric sphere Laser focus creates large E-field gradient Bead attracted to center of focus Want High NA for better trapping

8. Comparison of Unzipped DNA to Simulated Unzipping Simulated unzipping of pBR322 plasmid Simulated plasmid data hidden inside simulation of entire yeast genome Comparison with actual plasmid unzipping matches only the simulated plasmid data! A Correct Match, Score 0.2 18 Force (pN) 12 0 1500 Unzipping fork index (bp) B Mismatch, Score 0.8 18 Force (pN) 12 0 1500 Unzipping fork index (bp)

9. The Unzipping Construct We can use a similar setup for stretching DNA experiments as well…

10. Latest Results: Stretching DNA 3 4 2 Force 1 Distance dsDNA stretches with minimal effort Limits of DNA flexibility reached, hard to stretch, base-pairs break apart dsDNA overstretches – basically stretching two single strands of DNA DNA breaks or bead detaches from DNA

12. Attach bead to DNA