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Candidacy Exam Final Version

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This is a presentation I gave for my Candidacy for PhD. I present on the possibilities of probing protein-DNA interactions using Optical Tweezers. I discuss simulating force curves from optical …

This is a presentation I gave for my Candidacy for PhD. I present on the possibilities of probing protein-DNA interactions using Optical Tweezers. I discuss simulating force curves from optical tweezers, background information, and the molecular biological preparations involved. Finally I conclude with future applications of the technique that range from analysis of alternative splicing, transcriptional studies, and telomere mapping.

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Transcript

  • 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
    Me
    Bead Motility
    Making Kinesin
    60um
  • 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
    ~2m DNA length per cell
    ~2nm wide
    ~20um cell diameter
    ~10um nucleus diameter
    Chromosomes – structure for mitotic cells
    Chromatin – where everything happens
    Molecular Biology of the Cell
  • 7. Nucleosomes
    DNA wrapped in histone proteins
    Proteins:
    H2A
    H2B
    H3
    H4
    Form octamer
    Form stable tetramer
    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
    Deletions, inversions, insertions, and single base changes (SNPs)
    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. Forces from < 1 pN to 100s pN
    Length precision ~ 1 nm
    Thermal energy (kBT)
    4 pN – nm = 1/40 eV
    Kinesin 8 nm step, 6 pN stall
    (molecular motor)
    RNA Polymerase 0.3 nm step, 25 pN stall
    DNA Unzipping 15 pN
    Why Optical Tweezers?
  • 13. 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
  • 14. Examples of Single Molecule Analysis II
    Unzipping can detect nucleosomes
    nucleosome
  • 15. 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
  • 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. Unzipping Simulation
    Energy depends on:
    Energy of ssDNA (FJC)
    Energy of base-pairing (DNA)
    In order to get force vs unzipping index curve need:
    EFJC
    EDNA
  • 18. 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)
  • 19. Match Data
    32 unzipped plasmid data compared to library
    Each time the best match score was the plasmid simulated data
  • 20. How do we get real data?
  • 21. 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
  • 22. 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
  • 23. 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
  • 24.
  • 25. Our Tweezers
  • 26. How do we unzip DNA?
    • Create unzipping construct
    • 27. Create Shotgun fragment clones for single molecule analysis
    • 28. Attach pieces together and tether to cover slide
  • The Unzipping Construct
    Courtesy of Diego
  • 29. Restriction Enzymes
    REs recognize a specific sequence of DNA and cut the DNA at or near the site.
  • 30. 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:
  • 31. Ligating Construct to unzippable DNA
    Ligate – attach separate DNA strands into one continuous strand
    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?
  • 32. 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
  • 33. How Cloning Works
    Plasmids are:
    Extra chromosomal
    Capable of replication
    Useful for cloning
    Cloning is:
    Identical copying of fragment of DNA
    DNA can be inserted into plasmid for replication via Multiple Cloning site
    Wikipedia.org
    Fermentas.com
  • 34. Cloning
    LacZ gene turns cell blue
    Disrupting gene turns cells white
    Can select specific colonies
    Each colony contains different genomic fragment
    fragment
    Wikipedia
    No fragment
  • 35. 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
  • 36. DNA Tethering
    Create flow cell from double stick tape, slide and coverslip
    Flow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequencially
  • 37. What’s Next?
  • 38. Calibrate and Unzip
    Can unzip without calibration
    Messy data analysis
    Calibrate with stuck beads and free moving beads
    Then I can get GOOD unzipping data
    this can be real soon
  • 39. 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
  • 40. Transcriptional Studies
    RNA Pol II unzipping profile
    Has been achieved for RNA Polymerase I (E. coli)
    Pol II analysis during initiation, elongation, and termination
    Stalled Pol II in Elongation from collaborator (K. Adelman)
  • 41. 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
  • 42. 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.
  • 43. Can I do it all?
    Shotgun DNA Mapping
    Transcription Unzipping
    Collaborator ready and willing
    Foundations for Chromatin Mapping
    Which incorporates transcription
    Telomere Mapping is gravy
    Kinesin huge possibility (depending on funding)
  • 44. Thank You Everyone!
    sley
    Lab
    Toyoko and Cory too…
    …And my committee!
  • 45. 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
  • 46. Initial Studies
    Using PHO5 as “calibrator”
    PHO5 is promoter with 4 well know nucleosome positions
    We can show mapping works
  • 47. Unzipping Sensitivity
    Unzipping can detect:
    Insertions
    Deletions
    Inversions
    Seen Right – DNA sequence with deletion (black) compared with original sequence (red)
  • 48. 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)
  • 49. Trapping
    0
  • 50. Calibrating Trap Stiffness with free bead
    viscosity
    where
    radius of particle
    Power spectrum from
    Fourier t’form
    0, mass term insignificant in regime of frequency
  • 51. Profile from Stuck Bead(used in calibrating trap)
  • 52. Overview of Simulation
    The simulation is based on a quasi-equilibrium model. This is achieved by calculating the expectation values for Force and unzipping index.
    EFJC
    EDNA
    Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492
    Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.
  • 53. Overview of Simulation
    EDNAis the energy to break the base pairs.
    EFJC
    EDNA
    Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492
    Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.
  • 54. Overview of Simulation
    EFJC is the energy of single stranded DNA. As the dsDNA unzips this increases.
    EFJC
    EDNA
    Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Opening DNA with Piconewton Forces. Physical Review Letters , 4489-4492
    Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.