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