Download - Biotechnology basic techniques
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