DNA Tools

Biotechnology

Biotechnology

manipulation of biological organisms (usually with DNA itself)

Molecular Genetic TechniquesIsolating DNAAmplifying DNA

In vitro: Polymerase Chain reaction – Fig 20.7In vivo: Transformation & growth

Cutting DNARestriction enzyme digestion – Fig 20.2Application: Recombinant DNA

Visualizing DNASeparating DNA: Gel electrophoresis – Fig

20.8Staining DNA

DNA Isolation

Before DNA can be manipulated, it needs to be isolated from the cells.

DNA Isolation

Steps:1.Disrupt cell

membrane with a detergent

2.Precipitate DNA with ethanol

3.Retrieve precipitated DNA and store

http://www.cambio.co.uk/library/images/html_images/masterpure_complete_graph.gif

X

Resuspend DNA

Step 1

Step 2Step 3

DNA Amplification In vitro: PCR

Polymerase chain reactionPurpose: to increase the amount of DNA

DNA amplificationCreate millions of copies of a specific DNA

sequence syntheticallyDNA replication in a tube

Brainstorm

What are the major steps in DNA replication?

What substances do you need in each step of DNA replication?

Comparing DNA Replication

Steps Natural PCR

Unwinding

Ori on DNA templateHelicase

DNA templateHeat

Priming

RNA primerPrimase

DNA primer (2)Annealing temperature

ElongationNucleotidesDNAP

NucleotidesTaq polymerase

Termination

End of chromosome Meet another replication bubble

End of DNA templateChange in temperature

PCR Materials

DNA templateNucleotidesDNAP (Taq polymerase)DNA primers

PCR Cycle

1. Denaturation (95°C)

2. Primer annealing (50°C - 65°C)

3. Synthesis (72°C)

http://www.foodsafetywatch.com/public/images/1050a.jpg Fig. 20.7

PCR Animation

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

http://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.html (1: 27)

Show types of fragments created and counts their numbers (2:53)

http://www.youtube.com/watch?v=eEcy9k_KsDI

PCR

At which cycle do you get the correctly sized target sequence?

http://enfo.agt.bme.hu/drupal/sites/default/files/pcr_0.png

Primers

synthetic sequence single-stranded DNA (20-30 nucleotides)provides the needed 3’OH for

polymerization2 primers used complementary to each

end of the target region of the DNA

Primers identify target sequence

http://www.mun.ca/biology/scarr/PCR_detailed.jpg

What’s up with Taq?

Why can’t we use the DNAP that is found in most bacteria?

Taq Polymerase

DNAP Isolated from bacteria (Thermus aquaticus) that live in hot springs

Heat stable enzyme (can withstand extreme temperatures)

http://www.amath.washington.edu/~qian/TheScientist/TheScientists_files/geyser.jpg

Water baths to thermal cycler

Before taq3 water baths at 3

temperatures re-addition of DNAP

at the beginning of each synthesis step

http://www.molecularstation.com/molecular-biology-images/data/509/old-pcr-machine.JPG

PCR Efficiency

Thermal cycler:has a plate that

heats and coolsProcess takes 1–

2 hoursAfter 30 cycles,

230 (more than a billion) copies of DNA can be produced.

http://biolot.com/upload/12_1.jpg

Kary Mullis

Developed PCR in 1986Received Nobel Prize in 1993

Interviews Naming PCR

http://www.dnalc.org/view/15138-Naming-PCR.html Making many DNA copies

http://www.dnalc.org/view/15140-Making-many-DNA-copies-Kary-Mullis.html

Finding DNA to copy

http://www.dnalc.org/view/15139-Finding-DNA-to-copy-Kary-Mullis.html

Procedure and Rationale

Tutorialshttp://www.sumanasinc.com/webcontent/anima

tions/content/pcr.htmlhttp://www.dnalc.org/ddnalc/resources/pcr.html

PCR Virtual Labhttp://learn.genetics.utah.edu/content/labs/pcr/

Game & lecturehttp://nobelprize

.org/educational_games/chemistry/pcr/game/index.html

About 20 minutes long (with lecture)

DNA Amplification in vivo

Bacterial cells are transformed (uptake external DNA)

Bacteria are grown in a liquid mediumAs the total number of bacterial cells increase

the total amount of DNA will increase

http://www.viewingspace.com/genetics_culture/pages_genetics_culture/gc_w03/kac_webarchive/genesis_page/transformation/trans.jpg

Methods of Bacterial Transformationtreat bacteria to make cell walls

permeable to uptake genetic material Cold CaCl2 treatment followed by heat-

shockingmakes cell membrane leaky and more

permeableElectroporation

electric current to increase cell permeability

in vitro and in vivo DNA Amplification

System in vitro in vivo

Definition

DNA amplification

Advantage of system

in vitro and in vivo DNA Amplification

System in vitro in vivo

Definition Outside of natural environment

Inside natural environment (cellular)

DNA amplification

Advantage of system

in vitro and in vivo DNA Amplification

System in vitro in vivo

Definition Outside of natural environment

Inside natural environment (cellular)

DNA amplification

PCR Bacterial cell transformed with DNA

Advantage of system

in vitro and in vivo DNA Amplification

System in vitro in vivo

Definition Outside of natural environment

Inside natural environment (cellular)

DNA amplification

PCR Bacterial cell transformed with DNA

Advantage of system

DNA can be in a partially degraded state and still be amplified

few errors in replication due to proofreading [thus limitation to the number of times PCR cycle can be repeated]

Restriction Enzymes

Biological “scissors”Endonuclease:

break phosphodiester bonds within a nucleotide chain (as opposed to at the ends of a chain)

Fig. 20.2

Restriction Enzyme FunctionFound naturally in bacteria“immune system” of bacteria

protect bacteria against intruding DNA from other organisms (phages, other bacteria)

recognize short nucleotide sequences in the foreign DNA

cut covalent phosphodiester bonds of both strands of DNA, rendering foreign DNA harmless

Restriction Site

Each restriction enzyme has a specific sequence that it recognizes and a specific location on the sequence where it cuts

Restriction site: sequence recognized and cut by restriction enzyme

Characteristic of restriction site:4-8 bp in lengthPalindromic: same sequence on

complementary strand in opposite orientation

Fig. 20.2

5’ G A A T T C 3’3’ C T T A A G 5’

Restriction Enzyme Digestion

Enzyme cuts at the

same site on both strands

Restriction fragments:

pieces of DNA created by

restriction enzymes

Resulting fragment can have: Sticky End: a single-stranded end of the restriction

fragment Blunt ends: straight ends without any single-stranded

regionsFig. 20.2

Example: Sticky end with 5’ overhang5’ overhang with EcoRI digestion

5’ G A A T T C 3’3’ C T T A A G 5’

5’ G 3’ 5’ A A T T C 3’

3’ C T T A A 5’ 3’ G 5’

Example: Sticky end with 3’ overhang

3’ overhang with PstI digestion

5’ C T G C A G 3’3’ G A C G T C 5’

5’ C T G C A 3’ 5’ G 3’3’ G 5’ 3’ A C G T C 5’

Example: Blunt end

blunt ends with SmaI digestion

5’ C C C G G G 3’3’ G G G C C C 5’

5’ C C C 3’ 5’ G G G 3’3’ G G G 5’ 3’ C C C 5’

Restriction Enzyme Examples

http://barleyworld.org/css430_09/lecture%208-09/figure-11-04.JPG

Naming Restriction EnzymesRestriction enzymes are named

according to the organism from which it was identified.

Example: EcoRIE - Escherichiaco - coliR - strain RY13I - 1st enzyme in this strain

Practice: Naming Restriction EnzymesBacillus amyloliquefaciens, strain H, 5th

endonuclease identified

Nocardia otitidis, 3rd endonuclease identified

BamHV

NotIII

Animation: Restriction Enzyme

http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

Tutorial:

http://www.dnalc.org/resources/animations/restriction.html

Application of restriction enzymes

Any DNA cut with the same restriction can be ligated together because they have the same sticky ends that are complementary

http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg

Recombinant DNA

DNA in which genes from two different sources (often different species) are combined into one molecule

Usually the gene of interest is inserted into a bacterial plasmid

Why do you think a bacterial plasmid is used?

Fig. 20.3

Forming Recombinant DNA

Restriction enzyme digestion of plasmid and gene of interest

Hybridization of matching sticky ends on gene of interest and plasmid

DNA ligase seals gene of interest with plasmid

Activity: Recombinant DNAYou are given either a gene of interest

(linear) or a plasmid (circular)Cut out your DNADigest it with the given restriction enzymeFind the person with a matching sticky

end to form the recombinant

Additional: Research the data pair. Prepare a short (3-4 sentence) write-up that relates to the two terms.

Gel Electrophoresis

Size separation of nucleic acids by moving them through a gel medium using electric currentCan also be used for protein

Gel Electrophoresis

DNA is negatively charge (phosphates) thus will move toward the positive electrode

Making the gel

Liquid solutions of the gel medium is poured into a mould and allowed to set and solidify

Notice the comb is placed at one end of the gel. When it is removed it creates the wells where DNA is loaded.

Positive (+) electrode

Negative (-) electrode

Where DNA is loaded onto gel

Blue liquid is buffer

http://cg.scs.carleton.ca/~morin/teaching/compbio/electro.html

Horizontal Gel

Vertical Gel http://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg

Vertical Gel

Types of Gel Medium

Medium Agarose Polyacrylamide

Source Seaweed extract Artificial polymer

Resolving power

Lower resolution Higher resolution

Separation Nucleic acidNucleic acid Protein

Separation by Size

Gel medium is semi-solid and provides resistance for DNA movement.

The more concentrated the gel, the higher the resolving power of the gel.

Separation by Size

Short DNAmoves through gel easilytravels further

Long DNAmoves through gel slower because its size

gets caught in the web of polymers that make up a gel

does not move as far

http://cg.scs.carleton.ca/~morin/teaching/compbio/f_s02gelelect.gif

Other Materials

Liquid buffer containing ions:Provides a medium for the flow of electric

currentPrevents gel from overheating and drying

out.Coloured dyes mixed with DNA:

track distance travelled on the gel by DNAIncrease density of DNA so that it sinks to

bottom of wells

negativeelectrode

positiveelectrode

agarose gel

Loading dye

Loading dye

Animations: gel electrophoresis

http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

DNA Visualization from GelLoading dye doesn’t make DNA on the

gel visibleNeed to stain DNA to see it

DNA staining: Ethidium bromideA chemical that binds to the DNA

carcinogenic!!!Glows under UV light

Use UV light box to see fluorescent DNA bands

DNA stained with ethidum bromide exposed under UV light

Nerdy gifts