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

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Shotgun DNA Mapping for IGERT