candidacy exam final version
DESCRIPTION
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.TRANSCRIPT
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Shotgun DNA Mapping
Anthony Salvagno
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Welcome to KochLab!
Single Molecule DNA Analysis Kinesin Studies
F
F
Image from Block and adapted by Koch Image by Koch
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Kinesin Studies
• Andy– Gliding Motility Assay– Surface Passivation
• Larry– Tracking– Processivity
• Brigette– Ensemble ATP Hydrolysis
• Me– Bead Motility– Making Kinesin
60um
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Single Molecule DNA Studies
• What is DNA?• What is Shotgun DNA
Mapping?• What are Optical
Tweezers?• What is Molecular
Biology?
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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
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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
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Nucleosomes
• DNA wrapped in histone proteins
• Proteins:– H2A– H2B– H3– H4
• Form octamer
Wikipedia
Form stable tetramer
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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
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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
RNA Pol II
Transcription
Reassembled Nucleosomes
promoter
crypticpromoter
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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
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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
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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?
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Examples of Single Molecule Analysis
• Red Line – protein bound to DNA• Black Line – naked DNA• Black Dotted Line- predictions of
protein locations
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F
• Unzipping can detect proteins bound to DNA
Koch et al. 2002
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Examples of Single Molecule Analysis II
• Unzipping can detect nucleosomes
nucleosome
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Shotgun DNA Mapping
• Want to understand how proteins affect gene expression
• Need a way to map sequences of DNA to location in genome
Step 1: Digest genome into fragments
Step 2: Unzip fragment and record forces
Step 3: Compare experimental forces to a library of simulated curves
Genomic DNA
Endonuclease
dsDNA anchor
Random fragment
Experimental Force
Library of Simulated Curves
Correct Match
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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
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Unzipping Simulation
• Energy depends on:– Energy of ssDNA (FJC)– Energy of base-pairing
(DNA)
• In order to get force vs unzipping index curve need:
FJCDNA EEH
EFJC
EDNA
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Proof of Principle
• Simulated unzipping of pBR322 plasmid
• Simulation info hidden in genomic simulation
• Old unzipping data (Koch) used for comparison
12
18
Forc
e (p
N)
0 1500Unzipping fork index (bp)
Simulated data
Optical tweezersData (Koch 2002)
SimulationOT Data
Correct Match, Score 0.2A
0 1500Unzipping fork index (bp)
Simulated data
Optical tweezersData (Koch 2002)
OT DataSimulation
Mismatch, Score 0.8
12
18
Forc
e (p
N)
B
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Match Data
• 32 unzipped plasmid data compared to library
• Each time the best match score was the plasmid simulated data
File Number (Arb.)
Mat
ch Sc
ore
Match
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How do we get real data?
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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
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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
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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
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Our Tweezers
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How do we unzip DNA?
•Create unzipping construct•Create Shotgun fragment clones for single molecule analysis•Attach pieces together and tether to cover slide
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The Unzipping Construct
Courtesy of Diego
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RE
XhoI
EcoRI
NotI
SapI
BstXI
Recognition Sequence
CTCGAGGAGCTC
GAATTCCTTAAG
GCGGCCGCCGCCGGCG
GCTCTTCNNNNNCGAGAAGNNNNN
CCANNNNNNTGGGGTNNNNNNACC
Cut
C TCGAGGAGCT C
G AATTCCTTAA G
GC GGCCGCCGCCGG CG
GCTCTTCN NNNNCGAGAAGNNNN N
CCANNNNN NTGGGGTN NNNNNACC
Restriction Enzymes
• REs recognize a specific sequence of DNA and cut the DNA at or near the site.
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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
BstXI
GCTCTTCNNNNNCGAGAAGNNNNN
CCANNNNNNTGGGGTNNNNNNACC
GCTCTTCN NNNNCGAGAAGNNNN N
CCANNNNN NTGGGGTN NNNNNACC
Recall:
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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?
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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
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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
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Cloning
• LacZ gene turns cell blue
• Disrupting gene turns cells white
• Can select specific colonies – Each colony contains
different genomic fragment
Wikipedia
fragment
No fragment
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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
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DNA Tethering
• Create flow cell from double stick tape, slide and coverslip• Flow anti-dig, surface blocker, tethering DNA, microspheres,
and wash sequencially
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What’s Next?
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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
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Chromatin Studies
• Shotgun Chromatin Mapping– Can insert random
fragments into yeast to get chromatin
– Want to map nucleosome and protein locations
Optical Trap
ssDNA
Coverglass
nucleosome
Elongating Pol II
Koch
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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)
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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
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Telomere Studies
• Telomere mapping– Highly repetitive DNA– Not easily sequenced
• Telomerase structure• T-loops
This DNA Molecule has17 nearly identical~200 bp repeats
Koch
Griffin et al.
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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)
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Thank You Everyone!
…And my committee!
Toyoko and Cory too…
sley Lab
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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
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Initial Studies
• Using PHO5 as “calibrator”
• PHO5 is promoter with 4 well know nucleosome positions
• We can show mapping works
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Unzipping Sensitivity
• Unzipping can detect:– Insertions– Deletions– Inversions
• Seen Right – DNA sequence with deletion (black) compared with original sequence (red)
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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)
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Trapping
0
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222
2
2222222
)(
)()()()(
6
)(
k
BX
BFXXkm
a
tfkxxxm
where
Power spectrum fromFourier t’form
viscosity
radius of particle
0, mass term insignificant in regime of frequency
Calibrating Trap Stiffness with free bead
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Profile from Stuck Bead(used in calibrating trap)
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Overview of Simulation
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.
WLCFJCDNA EEEH
The simulation is based on a quasi-equilibrium model. This is achieved by calculating the expectation values for Force and unzipping index.
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Overview of Simulation
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.
WLCFJCDNA EEEH
EDNA is the energy to break the base pairs.
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Overview of Simulation
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.
WLCFJCDNA EEEH
EFJC is the energy of single stranded DNA. As the dsDNA unzips this increases.