A Thorough Introduction to Shotgun DNA Mapping and Kicking Ass in Science

By Anthony

Presents:

I made this…

…and this

Thank You Osley Lab

• A big thanks to Kelly and Mary Ann… • Thanks Cory and Toyoko…

…and KochLab

Motivation• Need better ways to study native chromatin• Single-molecule analysis can have a huge

impact• Kornberg said so:

“By pulling at the DNA with forces strong enough to unwrap DNA from the histone octamer, the optical tweezer allows for counting the remaining nucleosomes at the end of the remodeling process.”

RNA Pol II

Transcription

Reassembled Nucleosomes

promoter

crypticpromoter

Shotgun DNA Mapping in a NutshellProcedure

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

Austin is in there too

What this talk is mostly about

How will it work on native chromatin?

• Unzip with everything attached

• Allow dsDNA to rezip by relaxing strand

• Unzip naked DNA• Use matching program

to locate strand and binding sites in genome

dsDNA Bound protein etc.

How Matching Works

12

18

Forc

e (p

N)

0 1500Unzipping fork index (bp)

Simulated data

Optical tweezersData (Koch 2002)

SimulationOT Data

Correct Match, Score 0.2

0 1500Unzipping fork index (bp)

Simulated data

Optical tweezersData (Koch 2002)

OT DataSimulation

Mismatch, Score 0.8

12

18Fo

rce

(pN

)A B

•Using known physical models you can predict (simulate) what the force profile of unzipped dsDNA would look like•You then can compare that to actual unzipping data•Correct matches yield match scores close to zero, just how Larry defined it

More about matching

• When actual and simulated unzipping events are compared we can see one distinct match

• Test of 32 tethers of same sequence of unzipped DNA worked every time!

File Number (Arb.)

Mat

ch Sc

ore

Match

How do Tweezers Work?

We can measure forces on the bead based on deflectionsobserved by a QPD (quadrant photodiode).

Our Optical Tweezers

movie of tweezer setup

Where do you start?

• Need genomic DNA from yeast

• Grow some yeast• Extract the DNA• Now we’re Koching

A blurry image of yeast cells

Yeast Cell

• Spheroplasting• RNaseA-ing• Phenol/Chloroform

Extraction and Ethanol Precipitation

It’s foreign so it’s gotta be evil

Next Step

• Need digested plasmid DNA and digested genomic DNA

• Want to clone fragments– For sequencing– So we can unzip a lot of

fragmentsMichael Bay’s next film… too late I already sold the rights

The first of many gels

• Lanes:1: pRS413 uncut2: pRS cut with XhoI3: pRS cut with NotI4: pRS cut with BstXI5: genomic uncut DNA

(gDNA)6: gDNA cut with XhoI (didn’t work)

10kb length

My archnemesis

Digested gDNA

• Lanes:1: Uncut gDNA2: gDNA cut with XhoI3: gDNA cut with XhoI (for redundancy)

Making this was really annoying

Get used to this, there is a lot more coming

Inserting DNA

• CIP – Calf Intestinal Phosphatase

• T4 DNA Ligase - ??? DNA Ligase

Terminators can’t self terminate.

Making Clones

• Mix Competent E. coli cells with plasmid DNA

• E. coli readily replicates plasmid

• Grow cells on petri dish• Cells grow into

individual colonies• If plasmid has inserts

then each colony is a separate insert

One of them likes pizza

Transformation Success?

This is all Koch’s fault

E. Coli DNA

Extracted plasmid DNA

Double Digest and pBluescript

I was drunk when I took this pictureI was drunk when I slept with this one

Redoing with pBS

• Now that is definitely some random genomic fragments

• Top Image quick extraction

• Bottom Image is good extraction

I like pink tape

Sequencing

• Involves some steps I don’t know

• Need to sequence so that when we unzip we can know what the correct match is

• Larry look away

I thought it would be funny if I used a print screen of this slide for this slide.

Not for Larry

’s Eyes

Tether Construction

• Make an oligo that has BstXI site and is Biotinylated

• We made 2:– One is a hairpin with a NotI

site– The other is two single

stranded oligos with a SapI site

• Remember our fragments have both NotI ends and SapI ends

pRL fragmentBstXI

or

NotI

SapI

NotI hairpin

Top and bottomAnnealed oligos

NotI end

SapI end

The sequel to Michael Bay’s movieRights also already sold

When it’s all done

• More on next slide

…

gDNA plasmidBiotinylated fragmentDigitylated fragment

This is what skittles does to your DNAGel of Digested Fragments

The quality of this image is a direct result of a computer from 1991

What I have now

1991 strikes again

anchor

fragment

both

What it should look likeWhat it looks like

2009 artist rendition

Combine with Fragments

• Ligate the plasmid random fragments to the tethering construct

• Use flow chamber fluidics to prepare sample for tweezing

• Tweeze

The Future

• Unzipping Nucleosomes• Unzipping RNA Pol II from two directions

– Hope is that it will provide different unzipping signatures

No Mas