shotgun dna mapping for igert
DESCRIPTION
This is a talk I gave to the IGERT fellows discussing my project in ~15min.TRANSCRIPT
Shotgun DNA Mapping
Anthony Salvagno
Shotgun DNA Mapping
• Prove that we can map DNA fragments in genome• Unzipped DNA has a unique signature• Want to match unzipped DNA to library of simulated
unzipping curves
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
DNA: The Code of Life
• Double stranded flexible polymer
• Covalently bonded sugar molecules make up the backbone
• Hydrogen bonded bases join two strands of DNA
• There are 4 bases
Whyfiles.org
Why do we care about DNA?• DNA controls every aspect of
who we are!• Proteins bound to DNA can
explain a lot about their function– DNA compaction– Transcription– Mutations
• Looking at naked DNA can also diagnose potential problems– Deletions– inversions
Power 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
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
Comparison of Unzipped DNA to Simulated Unzipping
• Simulated unzipping of pBR322 plasmid
• Simulated plasmid data hidden inside simulation of entire yeast genome
• Comparison with actual plasmid unzipping matches only the simulated plasmid data!
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
The Unzipping Construct
We can use a similar setup for stretching DNA experiments as well…
Latest Results: Stretching DNA
1
2
3
1. dsDNA stretches with minimal effort2. Limits of DNA flexibility reached, hard to stretch, base-pairs break apart3. dsDNA overstretches – basically stretching two single strands of DNA4. DNA breaks or bead detaches from DNA
Forc
e
Distance
4
Coming Soon: DNA Unzipping
•Tether unzipping construct to glass•Attach bead to DNA•Pull with twezers
Thank You Everyone!
sley Lab