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Shotgun DNA Mapping<br />Anthony <br />Salvagno<br />
Welcome to KochLab!<br />Single Molecule DNA Analysis<br />Kinesin Studies<br />F<br />F<br />Image from Block and adapted...
Kinesin Studies<br />Andy<br />Gliding Motility Assay<br />Surface Passivation<br />Larry<br />Tracking<br />Processivity<...
Single Molecule DNA Studies<br />What is DNA?<br />What is Shotgun DNA Mapping?<br />What are Optical Tweezers?<br />What ...
DNA: The Code of Life<br />Double stranded polymer<br />Covalently bonded sugar molecules make up the backbone<br />Hydrog...
DNA Compaction<br />Lots of DNA in a genome that needs to fit in the nucleus<br />Chromosomes – what we see in a cell thro...
Nucleosomes<br />DNA wrapped in histone proteins<br />Proteins:<br />H2A<br />H2B<br />H3<br />H4<br />Form octamer<br />W...
From DNA to People<br />DNA to RNA to Proteins<br />Known as gene expression<br />Leads to changes in characteristics betw...
Transcription<br />RNA Polymerase II:<br />Copies single strand of DNA to make RNA <br />Moves with transcription bubble<b...
Points about Gene Expression<br />Mutations can affect many aspects of gene expression<br />Possible changes because of:<b...
Why Single Molecule is Powerful<br />Bulk studies provide general insight<br />Information is average from all molecules i...
Examples of Single Molecule Analysis<br />Red Line – protein bound to DNA<br />Black Line – naked DNA<br />Black Dotted Li...
Examples of Single Molecule Analysis II<br />Unzipping can detect nucleosomes<br />nucleosome<br />
Shotgun DNA Mapping<br />Want to understand how proteins affect gene expression<br />Need a way to map sequences of DNA to...
Unzipping Simulation<br />Energy depends on:<br />Energy of dsDNA (WLC)<br />Energy of ssDNA (FJC)<br />Energy of base-pai...
Unzipping Library<br />Used Yeast Genome because less complex than human, but can still have Chromatin<br />Simulated dige...
Proof of Principle<br />Simulated unzipping of pBR322 plasmid<br />Simulation info hidden in genomic simulation<br />Old u...
Match Data<br />32 unzipped plasmid data compared to library <br />Each time the best match score was the plasmid simulate...
How do we get real data?<br />
Optical Tweezers<br />Focused laser light has the ability to trap small particles<br />Simplest trap is composed of just a...
Optical Trap<br />Bead is tiny dielectric sphere<br />Laser focus creates large E-field gradient<br />Bead attracted to ce...
Data Collection<br />Refraction of laser from bead moves path<br />QPD tracks motion of beam<br />Force in trap approx. as...
Our Tweezers<br />
How do we unzip DNA?<br /><ul><li>Create unzipping construct
Create Shotgun fragment clones for single molecule analysis
Attach pieces together and tether to cover slide</li></li></ul><li>The Unzipping Construct<br />Courtesy of Diego<br />
Restriction Enzymes<br />REs recognize a specific sequence of DNA and cut the DNA at or near the site.<br />
Piece by Piece Construct Creation<br />Anchor<br />Made from PCR of pRL574<br />Has BstXI overhang with known base sequenc...
Ligating Construct to unzippable DNA<br />Adapter oligo contains nick and biotinylated DNA base<br />Need to ligate in spe...
Making Shotgun Clones<br />Why clone?<br />We can have a ton of a specific DNA fragment<br />Some for unzipping<br />Some ...
Yeast<br />Want pure genomic DNA<br />Need to get rid of:<br />Cell wall and membrane<br />Proteins<br />Organelles<br />N...
Genome Digestion<br />Need to make fragments from pure genomic DNA<br />XhoI digest produces very large fragments<br />Xho...
Cloning<br />Plasmids are circularized DNA with replication start sequence<br />pBluescript allows for blue/white selectio...
DNA Tethering<br />Create flow cell from double stick tape, slide and coverslip<br />Flow anti-dig, surface blocker, tethe...
What’s Next?<br />
Chromatin Studies<br />Shotgun Chromatin Mapping<br />Can insert random fragments into yeast to get chromatin<br />Want to...
Transcriptional Studies<br />RNA Pol II unzipping profile<br />Has been achieved for RNA Polymerase I (E. coli)<br />Pol I...
A Little About Telomeres<br />During Replication, ends of DNA are lost<br />Telomeric DNA caps ends to prevent disaster<br...
Telomere Studies<br />Telomere mapping<br />Highly repetitive DNA<br />Not easily sequenced<br />Telomerase structure<br /...
Thank You Everyone!<br />Toyoko and Cory too…<br />…And my committee!<br />
Gel Electrophoresis<br />Electric field applied to charged molecules<br />DNA is negatively charged<br />Gel lattice cause...
Initial Studies<br />Using PHO5 as “calibrator”<br />PHO5 is promoter with 4 well know nucleosome positions<br />We can sh...
Unzipping Sensitivity<br />Unzipping can detect:<br />Insertions<br />Deletions<br />Inversions<br />Seen Right – DNA sequ...
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Practice Candidacy Exam

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A practice talk about Shotgun DNA Mapping. I talk about gene expression, optical tweezers, unzipping DNA, mapping DNA fragments, telomeres, RNA transcription, and much more!

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Practice Candidacy Exam

  1. 1. Shotgun DNA Mapping<br />Anthony <br />Salvagno<br />
  2. 2. Welcome to KochLab!<br />Single Molecule DNA Analysis<br />Kinesin Studies<br />F<br />F<br />Image from Block and adapted by Koch<br />Image by Koch<br />
  3. 3. Kinesin Studies<br />Andy<br />Gliding Motility Assay<br />Surface Passivation<br />Larry<br />Tracking<br />Processivity<br />Brigette<br />Ensemble ATP Hydrolysis<br />Future<br />Bead Motility<br />
  4. 4. Single Molecule DNA Studies<br />What is DNA?<br />What is Shotgun DNA Mapping?<br />What are Optical Tweezers?<br />What is Molecular Biology?<br />
  5. 5. DNA: The Code of Life<br />Double stranded 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 />
  6. 6. DNA Compaction<br />Lots of DNA in a genome that needs to fit in the nucleus<br />Chromosomes – what we see in a cell through a microscope<br />Chromatin – where everything happens<br />Molecular Biology of the Cell<br />
  7. 7. Nucleosomes<br />DNA wrapped in histone proteins<br />Proteins:<br />H2A<br />H2B<br />H3<br />H4<br />Form octamer<br />Wikipedia<br />
  8. 8. From DNA to People<br />DNA to RNA to Proteins<br />Known as gene expression<br />Leads to changes in characteristics between organisms<br />Leads to differentiation amongst cell lines<br />Wikipedia<br />Thinkquest.org<br />
  9. 9. Transcription<br />RNA Polymerase II:<br />Copies single strand of DNA to make RNA <br />Moves with transcription bubble<br />Initiation<br />RNAPII assembly<br />Elongation<br />Active transcription<br />Termination<br />RNAPII disassembly<br />Reassembled Nucleosomes<br />RNA Pol II<br />promoter<br />cryptic<br />promoter<br />Transcription<br />
  10. 10. Points about Gene Expression<br />Mutations can affect many aspects of gene expression<br />Possible changes because of:<br />DNA sequence modifications<br />Post Translational Modifications<br />
  11. 11. Why Single Molecule is Powerful<br />Bulk studies provide general insight<br />Information is average from all molecules in sample<br />Different molecules have different properties<br />Studying DNA one molecule at a time can provide unprecedented understanding of a process<br />
  12. 12. Examples 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 />
  13. 13. Examples of Single Molecule Analysis II<br />Unzipping can detect nucleosomes<br />nucleosome<br />
  14. 14. Shotgun DNA Mapping<br />Want to understand how proteins affect gene expression<br />Need a way to map sequences of DNA to location in genome<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 />
  15. 15. Unzipping Simulation<br />Energy depends on:<br />Energy of dsDNA (WLC)<br />Energy of ssDNA (FJC)<br />Energy of base-pairing (DNA)<br />EFJC<br />EDNA<br />
  16. 16. Unzipping Library<br />Used Yeast Genome because less complex than human, but can still have Chromatin<br />Simulated digestion with XhoI<br />Over 1300 fragments<br />Simulated unzipping 2000bp before and after recognition sequence<br />Gives us over 2600 unzipping profiles<br />Unzipping Direction<br />
  17. 17. Proof of Principle<br />Simulated unzipping of pBR322 plasmid<br />Simulation info hidden in genomic simulation<br />Old unzipping data (Koch) used for comparison<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 />
  18. 18. Match Data<br />32 unzipped plasmid data compared to library <br />Each time the best match score was the plasmid simulated data<br />
  19. 19. How do we get real data?<br />
  20. 20. Optical Tweezers<br />Focused laser light has the ability to trap small particles<br />Simplest trap is composed of just a laser and an objective<br />SM Block<br />
  21. 21. Optical Trap<br />Bead is tiny dielectric sphere<br />Laser focus creates large E-field gradient<br />Bead attracted to center of focus<br />
  22. 22. Data Collection<br />Refraction of laser from bead moves path<br />QPD tracks motion of beam<br />Force in trap approx. as spring<br />F=-kx<br />La Porta Lab<br />
  23. 23. Our Tweezers<br />
  24. 24. How do we unzip DNA?<br /><ul><li>Create unzipping construct
  25. 25. Create Shotgun fragment clones for single molecule analysis
  26. 26. Attach pieces together and tether to cover slide</li></li></ul><li>The Unzipping Construct<br />Courtesy of Diego<br />
  27. 27. Restriction Enzymes<br />REs recognize a specific sequence of DNA and cut the DNA at or near the site.<br />
  28. 28. Piece by Piece Construct Creation<br />Anchor<br />Made from PCR of pRL574<br />Has BstXI overhang with known base sequence<br />Beginning of polymer is labeled with dig molecule for specific binding with anti-dig<br />Adapter<br />Short duplex made 2 single-stranded oligos<br />5’ end has phosphate removed creating a nick<br />5’ end has complementary BstXI overhang<br />3’ end has SapI/EarI overhang<br />SapI<br />GCTCTTCNNNNN<br />CGAGAAGNNNNN<br />GCTCTTCN NNNN<br />CGAGAAGNNNN N<br />BstXI<br />CCANNNNNNTGG<br />GGTNNNNNNACC<br />CCANNNNN NTGG<br />GGTN NNNNNACC<br />Recall:<br />
  29. 29. Ligating Construct to unzippable DNA<br />Adapter oligo contains nick and biotinylated DNA base<br />Need to ligate in specific way<br />Limited by genomic DNA<br />Low adapter duplex concentration, but gradually increase during the course of the reaction<br />Where does unzippable DNA come from?<br />
  30. 30. Making Shotgun Clones<br />Why clone?<br />We can have a ton of a specific DNA fragment<br />Some for unzipping<br />Some for sequencing<br />What is shotgunning?<br />Drinking a beer really fast<br />Creating random fragments quickly<br />
  31. 31. Yeast<br />Want pure genomic DNA<br />Need to get rid of:<br />Cell wall and membrane<br />Proteins<br />Organelles<br />Nuclear membrane<br />RNA<br />RPI.edu<br />
  32. 32. Genome Digestion<br />Need to make fragments from pure genomic DNA<br />XhoI digest produces very large fragments<br />XhoI+EcoRI provides much smaller fragment sizes<br />Need smaller fragments for cloning<br />
  33. 33. Cloning<br />Plasmids are circularized DNA with replication start sequence<br />pBluescript allows for blue/white selection<br />Each colony contains different fragment of DNA <br />Each cell in colony has the same fragment<br />Wikipedia<br />Fermentas.com<br />
  34. 34. DNA Tethering<br />Create flow cell from double stick tape, slide and coverslip<br />Flow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequencially<br />
  35. 35. What’s Next?<br />
  36. 36. Chromatin Studies<br />Shotgun Chromatin Mapping<br />Can insert random fragments into yeast to get chromatin<br />Want to map nucleosome and protein locations<br />Optical Trap<br />nucleosome<br />Elongating Pol II<br />ssDNA<br />Coverglass<br />Koch<br />
  37. 37. Transcriptional Studies<br />RNA Pol II unzipping profile<br />Has been achieved for RNA Polymerase I (E. coli)<br />Pol II analysis during initiation, elongation, and termination<br />
  38. 38. A Little About Telomeres<br />During Replication, ends of DNA are lost<br />Telomeric DNA caps ends to prevent disaster<br />Telomerase makes new telomere DNA from short RNA template<br />Wikipedia<br />
  39. 39. Telomere Studies<br />Telomere mapping<br />Highly repetitive DNA<br />Not easily sequenced<br />Telomerase structure<br />T-loops<br />This DNA Molecule has<br />17 nearly identical<br />~200 bp repeats<br />Koch<br />Griffin et al.<br />
  40. 40. Thank You Everyone!<br />Toyoko and Cory too…<br />…And my committee!<br />
  41. 41. Gel Electrophoresis<br />Electric field applied to charged molecules<br />DNA is negatively charged<br />Gel lattice causes smaller particles to travel faster than larger ones<br />Staining allows visualization of DNA<br />Direction of <br />DNA motion<br />
  42. 42. Initial Studies<br />Using PHO5 as “calibrator”<br />PHO5 is promoter with 4 well know nucleosome positions<br />We can show mapping works<br />
  43. 43. Unzipping Sensitivity<br />Unzipping can detect:<br />Insertions<br />Deletions<br />Inversions<br />Seen Right – DNA sequence with deletion (black) compared with original sequence (red)<br />
  44. 44. Polymerase Chain Reaction<br />Needed to make anchor<br />Start with template DNA and primers<br />Taq polymerase replicates DNA from primer location<br />Undergoes multiple cycles of melting, annealing, and replicating (extension)<br />For anchor one primer has dig molecule attached (digitylated)<br />

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