Download - Practice Candidacy Exam
Shotgun DNA Mapping
Anthony Salvagno
Welcome to KochLab!
Single Molecule DNA Analysis Kinesin Studies
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Image from Block and adapted by Koch Image by Koch
Kinesin Studies
• Andy– Gliding Motility Assay– Surface Passivation
• Larry– Tracking– Processivity
• Brigette– Ensemble ATP Hydrolysis
• Future– Bead Motility
Single Molecule DNA Studies
• What is DNA?• What is Shotgun DNA
Mapping?• What are Optical
Tweezers?• What is Molecular
Biology?
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
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
Nucleosomes
• DNA wrapped in histone proteins
• Proteins:– H2A– H2B– H3– H4
• Form octamer
Wikipedia
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
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
Points about Gene Expression
• Mutations can affect many aspects of gene expression
• Possible changes because of:– DNA sequence
modifications– Post Translational
Modifications
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
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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• Unzipping can detect proteins bound to DNA
Koch et al. 2002
Examples of Single Molecule Analysis II
• Unzipping can detect nucleosomes
nucleosome
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
Unzipping Simulation
• Energy depends on:– Energy of dsDNA (WLC)– Energy of ssDNA (FJC)– Energy of base-pairing
(DNA)
WLCFJCDNA EEEH
EFJC
EDNA
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
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
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
How do we get real data?
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
Optical Trap
• Bead is tiny dielectric sphere
• Laser focus creates large E-field gradient
• Bead attracted to center of focus
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
Our Tweezers
How do we unzip DNA?
•Create unzipping construct•Create Shotgun fragment clones for single molecule analysis•Attach pieces together and tether to cover slide
The Unzipping Construct
Courtesy of Diego
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.
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:
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?
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
Yeast
• Want pure genomic DNA
• Need to get rid of:– Cell wall and membrane– Proteins– Organelles– Nuclear membrane– RNA
RPI.edu
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
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
DNA Tethering
• Create flow cell from double stick tape, slide and coverslip• Flow anti-dig, surface blocker, tethering DNA, microspheres,
and wash sequencially
What’s Next?
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
Transcriptional Studies
• RNA Pol II unzipping profile– Has been achieved for RNA Polymerase I (E. coli)
• Pol II analysis during initiation, elongation, and termination
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
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.
Thank You Everyone!
…And my committee!
Toyoko and Cory too…
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
Initial Studies
• Using PHO5 as “calibrator”
• PHO5 is promoter with 4 well know nucleosome positions
• We can show mapping works
Unzipping Sensitivity
• Unzipping can detect:– Insertions– Deletions– Inversions
• Seen Right – DNA sequence with deletion (black) compared with original sequence (red)
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)