BIOTECHNOLOGYWhat can we do with DNA?

Biotechnology

• Manipulation of biological organisms or their components for research and industrial purpose

• Usually manipulate DNA itself

How to study individual gene? • To study the function of individual genes, molecular

biologists will cut them out of a genome and place them into bacteria

• Why study gene in bacteria?

Basic techniques1. DNA isolation2. Restriction enzyme digest3. DNA amplification

• Transformation and growth – In vivo• Polymerase Chain Reaction (PCR) – In vitro

4. Gel electrophoresis

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

1.Disrupt cell membranes with a detergent• Example of detergent: SDS, Tween-20

2.Precipitate DNA with ethanol3.Obtain precipitated DNA and storage

DNA Isolation

DNA isolation

How do you get specific sequence from the entire genome?

Restriction Enzyme Digestion• DNA must be cut into smaller pieces before they can be

used in other techniques.

Restriction Enzymes• Molecular scissors

• Restriction Endonucleases: digestive enzymes that recognize specific DNA sequences (known as Recognition site) and cut at specific points

Restriction Site• Typically 4-8 bp in length• Double-stranded DNA• Always palindromic:

What does palindromic mean?

5’ G A A T T C 3’3’ C T T A A G 5’

EcoRI recognition site

Same sequence on complementary strand in opposite orientation

Why use these enzyme?• Restriction enzymes are naturally found in bacteria• Restriction enzymes act as “immune system” of bacteria

• Protect bacteria against DNA from other organisms (ex. bacteriophage (bacterial virus))

• Recognize and cut phosphodiester bonds of foreign DNA, not its own genome -> making foreign DNA harmless for the cell

Restriction enzyme digestion• Restriction enzyme recognize a palindromic DNA

sequence in double-stranded DNA and cleave both strands

• Resulting Sticky End: a single-stranded overhangs• Sticky ends with 5’ overhang• Sticky ends with 3’ overhang• Blunt ends

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’

5’ overhang

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’

3’ overhang

blunt ends – enzyme digests to make straight ends

SmaI 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’

DNA LigaseT4 DNA ligase – used to chemically join sticky ends of DNA

together

Recombinant DNA

Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA

Restriction Enzyme Animation• http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

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

DNA AmplificationTransformation & growth PCR

Amplification of DNA (in vivo)Transformation & Growth• Treat bacteria to

make cell walls permeable to uptake of foreign DNA

• Transformed bacterial cell grow and divide to amplify DNA

What is transformation?

Amplification of DNA (in vitro)PCR

PCR = Polymerase Chain Reaction•Powerful technique to produce millions of copies of specific DNA.

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Successful PCR reaction1) Need something to replicate and someplace to start

1) Need something to open DNA and unwind it• AND something to stabilize it once unwound

1) Need something to provide the primer to initiate synthesis

2) Need something to synthesize the new DNA

• What you need:• Template DNA• dNTPs – nucleotides (dATP, dTTP, dCTP, dGTP)• Two specific Primers: short pieces (20-30 nucleotides) of

synthetic single-stranded DNA• First is complementary to one DNA strand at the beginning of the target

region• Second is complementary to opposite DNA stand at the end of the

targeted region• DNA polymerase –Taq polymerase

Successful PCR reaction1) Need something to replicate and someplace to start

1) Need something to open DNA and unwind it• AND something to stabilize it once unwound

1) Need something to provide the primer to initiate synthesis

2) Need something to synthesize the new DNA

Template DNA(chromosome, plasmid, etc) You can chose

Heat

You can chose the primers, and therefore specify EXACTLY what you want to amplify

DNA polymerase (Must be heat stable)*Taq polymerase

Taq polymerase• Isolated from Thermophilus aquaticus bacterium• These bacteria live in hot springs and has heat stable

enzyme that can withstand extreme temperatures

3 steps for each PCR cycle1. DNA strand denaturation (95 oC)

• Separate double strand DNA• Each strand becomes template strand

2. Primer annealing (50 – 65 oC)• Short DNA pieces bind to temperate strands

3. DNA strand synthesis (72 oC)• Produce new DNA strands

**

2 copies of targeted sequence after 3rd cycle

Power of PCR• After 30 cycles, 230 (more than a billion) copies of DNA

can be produced• 30 cycles of PCR takes ~1-2 hours to complete

- PCR is preformed using thermal cycler

PCR animation• http://www.sumanasinc.com/webcontent/animations/conte

nt/pcr.html

• 3D animation:• http://www.dnalc.org/view/15475-The-cycles-of-the-polym

erase-chain-reaction-PCR-3D-animation-with-no-audio.html

Gel ElectrophoresisSeparating DNA sequence

Gel Electrophoresis• Separate DNA through a gel medium using an electric

current

Why can we move DNA with electricity?• DNA has a negative charge. Therefore it will move toward

positive electrode

Gel Medium

The gel medium can be made from:1. agarose - seaweed extract2. polyacrylamide - artificial polymer

• The type of gel used is dependent on how well separated the DNA pieces need to be.

• Polyacrylamide has higher resolution than agarose.

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

Loading DyeDNA is colourless.

How do you know that it gets into the gel?• Coloured dyes are mixed with DNA to track distance

travelled – Loading Dye

negativeelectrode

positiveelectrode

agarose gel

Separation DNA by size

The gel provides resistance for DNA movement.

Short DNA• moves through gel easily• travels further in a set amount of time

Long DNA• requires more effort to move through gel• does not move as far in a set amount of time

Visualizing GelDNA is colourless.

How do you see where the DNA is after the separation is complete?

• DNA is stained with ethidium bromide• UV light box is used to see fluorescent DNA bands

Ethidium Bromide