BIOTECHNOLOGY What can we do with DNA?. Biotechnology Manipulation of biological organisms or their...
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Transcript of BIOTECHNOLOGY What can we do with DNA?. Biotechnology Manipulation of biological organisms or their...
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BIOTECHNOLOGYWhat can we do with DNA?
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Biotechnology
• Manipulation of biological organisms or their components for research and industrial purpose
• Usually manipulate DNA itself
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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?
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Basic techniques
1. DNA isolation
2. Restriction enzyme digest
3. DNA amplification• Transformation and growth – In vivo• Polymerase Chain Reaction (PCR) – In vitro
4. Gel electrophoresis
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DNA Isolation
Before 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 ethanol
3.Obtain precipitated DNA and storage
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DNA Isolation
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DNA isolation
How do you get specific sequence from the entire genome?
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Restriction Enzyme Digestion• DNA must be cut into smaller pieces before they can be
used in other techniques.
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Restriction Enzymes• Molecular scissors
• Restriction Endonucleases: digestive enzymes that recognize specific DNA sequences (known as Recognition site) and cut at specific points
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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
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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
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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
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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
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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
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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’
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DNA LigaseT4 DNA ligase – used to chemically join sticky ends of DNA
together
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Recombinant DNA
Complementary sticky ends from different pieces of DNA can be joined together – recombinant DNA
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Restriction Enzyme Animation• http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
• Tutotial:• http://www.dnalc.org/resources/animations/restriction.html
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DNA AmplificationTransformation & growth
PCR
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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?
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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 reaction
1) 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
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• 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
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Successful PCR reaction
1) 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
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Taq polymerase• Isolated from Thermophilus aquaticus bacterium• These bacteria live in hot springs and has heat stable
enzyme that can withstand extreme temperatures
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3 steps for each PCR cycle
1. 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
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**
2 copies of targeted sequence after 3rd cycle
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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
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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
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Gel ElectrophoresisSeparating DNA sequence
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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
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Gel Medium
The gel medium can be made from:
1. agarose - seaweed extract
2. 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.
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Liquid solutions of the gel is poured into a mould and allowed to set and solidify.
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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
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negativeelectrode
positiveelectrode
agarose gel
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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
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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
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Ethidium Bromide
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