1. Shotgun DNA Mapping Anthony Salvagno

2. Welcome to KochLab! Single Molecule DNA Analysis Kinesin Studies F F Image from Block and adapted by Koch Image by Koch

3. Kinesin Studies Andy Gliding Motility Assay Surface Passivation Larry Tracking Processivity Brigette Ensemble ATP Hydrolysis Future Bead Motility

4. Single Molecule DNA Studies What is DNA? What is Shotgun DNA Mapping? What are Optical Tweezers? What is Molecular Biology?

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

6. DNA Compaction Lots of DNA in a genome that needs to fit in the nucleus Chromosomes – what we see in a cell through a microscope Chromatin – where everything happens Molecular Biology of the Cell

7. Nucleosomes DNA wrapped in histone proteins Proteins: H2A H2B H3 H4 Form octamer Wikipedia

8. From DNA to People DNA to RNA to Proteins Known as gene expression Leads to changes in characteristics between organisms Leads to differentiation amongst cell lines Wikipedia Thinkquest.org

9. Transcription RNA Polymerase II: Copies single strand of DNA to make RNA Moves with transcription bubble Initiation RNAPII assembly Elongation Active transcription Termination RNAPII disassembly Reassembled Nucleosomes RNA Pol II promoter cryptic promoter Transcription

10. Points about Gene Expression Mutations can affect many aspects of gene expression Possible changes because of: DNA sequence modifications Post Translational Modifications

11. Why Single Molecule is Powerful Bulk studies provide general insight Information is average from all molecules in sample Different molecules have different properties Studying DNA one molecule at a time can provide unprecedented understanding of a process

12. Examples 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

13. Examples of Single Molecule Analysis II Unzipping can detect nucleosomes nucleosome

14. Shotgun DNA Mapping Want to understand how proteins affect gene expression Need a way to map sequences of DNA to location in genome 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

15. Unzipping Simulation Energy depends on: Energy of dsDNA (WLC) Energy of ssDNA (FJC) Energy of base-pairing (DNA) EFJC EDNA

16. Unzipping Library Used Yeast Genome because less complex than human, but can still have Chromatin Simulated digestion with XhoI Over 1300 fragments Simulated unzipping 2000bp before and after recognition sequence Gives us over 2600 unzipping profiles Unzipping Direction

17. Proof of Principle Simulated unzipping of pBR322 plasmid Simulation info hidden in genomic simulation Old unzipping data (Koch) used for comparison 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)

18. Match Data 32 unzipped plasmid data compared to library Each time the best match score was the plasmid simulated data

19. How do we get real data?

20. Optical Tweezers Focused laser light has the ability to trap small particles Simplest trap is composed of just a laser and an objective SM Block

21. Optical Trap Bead is tiny dielectric sphere Laser focus creates large E-field gradient Bead attracted to center of focus

22. Data Collection Refraction of laser from bead moves path QPD tracks motion of beam Force in trap approx. as spring F=-kx La Porta Lab

23. Our Tweezers

25. Create Shotgun fragment clones for single molecule analysis

27. Restriction Enzymes REs recognize a specific sequence of DNA and cut the DNA at or near the site.

28. Piece by Piece Construct Creation Anchor Made from PCR of pRL574 Has BstXI overhang with known base sequence Beginning of polymer is labeled with dig molecule for specific binding with anti-dig Adapter Short duplex made 2 single-stranded oligos 5’ end has phosphate removed creating a nick 5’ end has complementary BstXI overhang 3’ end has SapI/EarI overhang SapI GCTCTTCNNNNN CGAGAAGNNNNN GCTCTTCN NNNN CGAGAAGNNNN N BstXI CCANNNNNNTGG GGTNNNNNNACC CCANNNNN NTGG GGTN NNNNNACC Recall:

29. Ligating Construct to unzippable DNA Adapter oligo contains nick and biotinylated DNA base Need to ligate in specific way Limited by genomic DNA Low adapter duplex concentration, but gradually increase during the course of the reaction Where does unzippable DNA come from?

30. Making Shotgun Clones Why clone? We can have a ton of a specific DNA fragment Some for unzipping Some for sequencing What is shotgunning? Drinking a beer really fast Creating random fragments quickly

31. Yeast Want pure genomic DNA Need to get rid of: Cell wall and membrane Proteins Organelles Nuclear membrane RNA RPI.edu

32. Genome Digestion Need to make fragments from pure genomic DNA XhoI digest produces very large fragments XhoI+EcoRI provides much smaller fragment sizes Need smaller fragments for cloning

33. Cloning Plasmids are circularized DNA with replication start sequence pBluescript allows for blue/white selection Each colony contains different fragment of DNA Each cell in colony has the same fragment Wikipedia Fermentas.com

34. DNA Tethering Create flow cell from double stick tape, slide and coverslip Flow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequencially

35. What’s Next?

36. Chromatin Studies Shotgun Chromatin Mapping Can insert random fragments into yeast to get chromatin Want to map nucleosome and protein locations Optical Trap nucleosome Elongating Pol II ssDNA Coverglass Koch

37. Transcriptional Studies RNA Pol II unzipping profile Has been achieved for RNA Polymerase I (E. coli) Pol II analysis during initiation, elongation, and termination

38. A Little About Telomeres During Replication, ends of DNA are lost Telomeric DNA caps ends to prevent disaster Telomerase makes new telomere DNA from short RNA template Wikipedia

39. Telomere Studies Telomere mapping Highly repetitive DNA Not easily sequenced Telomerase structure T-loops This DNA Molecule has 17 nearly identical ~200 bp repeats Koch Griffin et al.

40. Thank You Everyone! Toyoko and Cory too… …And my committee!

41. Gel Electrophoresis Electric field applied to charged molecules DNA is negatively charged Gel lattice causes smaller particles to travel faster than larger ones Staining allows visualization of DNA Direction of DNA motion

42. Initial Studies Using PHO5 as “calibrator” PHO5 is promoter with 4 well know nucleosome positions We can show mapping works

43. Unzipping Sensitivity Unzipping can detect: Insertions Deletions Inversions Seen Right – DNA sequence with deletion (black) compared with original sequence (red)

44. Polymerase Chain Reaction Needed to make anchor Start with template DNA and primers Taq polymerase replicates DNA from primer location Undergoes multiple cycles of melting, annealing, and replicating (extension) For anchor one primer has dig molecule attached (digitylated)