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

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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

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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)