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Biotechnology basic techniques

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  • Recombinant DNA = DNA in which genes from two different sources (often different species) are combined in vitro into the same molecule Central to genetic engineering Does not occur in nature (not related to bacterial plasmids entering chromosomes)
  • Transcript

    • 1. DNAToolsBiotechnology
    • 2. Biotechnologymanipulation ofbiological organisms(usually with DNAitself)
    • 3. Molecular Genetic TechniquesIsolating DNAAmplifying DNAIn vitro: Polymerase Chain reaction – Fig 20.7In vivo: Transformation & growthCutting DNARestriction enzyme digestion – Fig 20.2Application: Recombinant DNAVisualizing DNASeparating DNA: Gel electrophoresis – Fig 20.8Staining DNA
    • 4. DNA IsolationBefore DNA can bemanipulated, it needs tobe isolated from thecells.
    • 5. DNA IsolationSteps:1. Disrupt cellmembrane with adetergent2.Precipitate DNAwith ethanol3.Retrieveprecipitated DNAand storehttp://www.cambio.co.uk/library/images/html_images/masterpure_complete_graph.gifXResuspendDNAStep 1Step 2Step 3
    • 6. DNA Amplification In vitro: PCRPolymerase chain reactionPurpose: to increase the amount of DNADNA amplificationCreate millions of copies of a specific DNA sequencesyntheticallyDNA replication in a tube
    • 7. BrainstormWhat are the major steps in DNA replication?What substances do you need in each step of DNAreplication?
    • 8. Comparing DNA ReplicationSteps Natural PCRUnwindingOri on DNA templateHelicaseDNA templateHeatPrimingRNA primerPrimaseDNA primer (2)Annealing temperatureElongationNucleotidesDNAPNucleotidesTaq polymeraseTerminationEnd of chromosomeMeet anotherreplication bubbleEnd of DNA templateChange in temperature
    • 9. PCR MaterialsDNA templateNucleotidesDNAP (Taq polymerase)DNA primers
    • 10. PCR Cycle1. 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
    • 11. 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 theirnumbers (2:53)http://www.youtube.com/watch?v=eEcy9k_KsDI
    • 12. PCRAt which cycle do you get thecorrectly sized target sequence?http://enfo.agt.bme.hu/drupal/sites/default/files/pcr_0.png
    • 13. Primerssynthetic sequencesingle-stranded DNA (20-30 nucleotides)provides the needed 3’OH for polymerization2 primers used complementary to each end of thetarget region of the DNA
    • 14. Primers identify target sequencehttp://www.mun.ca/biology/scarr/PCR_detailed.jpg
    • 15. What’s up with Taq?Why can’t we use the DNAP that is found in mostbacteria?
    • 16. Taq PolymeraseDNAP Isolated from bacteria(Thermus aquaticus) that live inhot springsHeat stable enzyme (canwithstand extremetemperatures)http://www.amath.washington.edu/~qian/TheScientist/TheScientists_files/geyser.jpg
    • 17. Water baths to thermal cyclerBefore taq3 water baths at 3temperaturesre-addition of DNAP atthe beginning of eachsynthesis stephttp://www.molecularstation.com/molecular-biology-images/data/509/old-pcr-machine.JPG
    • 18. PCR EfficiencyThermal cycler:has a plate thatheats and coolsProcess takes 1–2hoursAfter 30 cycles, 230(more than abillion) copies ofDNA can beproduced.http://biolot.com/upload/12_1.jpg
    • 19. Kary MullisDeveloped PCR in 1986Received Nobel Prize in 1993Interviews Naming PCRhttp://www.dnalc.org/view/15138-Naming-PCR.html Making many DNA copieshttp://www.dnalc.org/view/15140-Making-many-DNA-copies-Kary-Mullis.html Finding DNA to copyhttp://www.dnalc.org/view/15139-Finding-DNA-to-copy-Kary-Mullis.html
    • 20. Procedure and RationaleTutorialshttp://www.sumanasinc.com/webcontent/animations/content/pcr.htmlhttp://www.dnalc.org/ddnalc/resources/pcr.htmlPCR Virtual Labhttp://learn.genetics.utah.edu/content/labs/pcr/Game & lecturehttp://nobelprize.org/educational_games/chemistry/pcr/game/index.htmlAbout 20 minutes long (with lecture)
    • 21. DNA Amplification in vivoBacterial cells are transformed (uptake externalDNA)Bacteria are grown in a liquid mediumAs the total number of bacterial cells increase thetotal amount of DNA will increasehttp://www.viewingspace.com/genetics_culture/pages_genetics_culture/gc_w03/kac_webarchive/genesis_page/transformation/trans.jpg
    • 22. Methods of BacterialTransformationtreat bacteria to make cell walls permeable touptake genetic materialCold CaCl2 treatment followed by heat-shockingmakes cell membrane leaky and more permeableElectroporationelectric current to increase cell permeability
    • 23. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinitionDNAamplificationAdvantage ofsystem
    • 24. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationAdvantage ofsystem
    • 25. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationPCR Bacterial cell transformedwith DNAAdvantage ofsystem
    • 26. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationPCR Bacterial cell transformedwith DNAAdvantage ofsystemDNA can be in apartially degradedstate and still beamplifiedfew errors in replicationdue to proofreading [thuslimitation to the number of timesPCR cycle can be repeated]
    • 27. Restriction EnzymesBiological “scissors”Endonuclease: breakphosphodiester bondswithin a nucleotide chain(as opposed to at theends of a chain)Fig. 20.2
    • 28. Restriction Enzyme FunctionFound naturally in bacteria“immune system” of bacteriaprotect bacteria against intruding DNA from otherorganisms (phages, other bacteria)recognize short nucleotide sequences in the foreignDNAcut covalent phosphodiester bonds of both strandsof DNA, rendering foreign DNA harmless
    • 29. Restriction SiteEach restriction enzyme has a specific sequencethat it recognizes and a specific location on thesequence where it cutsRestriction site: sequence recognized and cut byrestriction enzymeCharacteristic of restriction site:4-8 bp in lengthPalindromic: same sequence on complementarystrand in opposite orientationFig. 20.25’ G A A T T C 3’3’ C T T A A G 5’
    • 30. Restriction Enzyme Digestion Enzyme cuts at thesame site on both strands Restriction fragments:pieces of DNA created byrestriction 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
    • 31. Example: Sticky end with 5’overhang5’ overhang with EcoRI digestion5’ 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’
    • 32. Example: Sticky end with 3’overhang3’ overhang with PstI digestion5’ 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’
    • 33. Example: Blunt endblunt ends with SmaI digestion5’ 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’
    • 34. Restriction Enzyme Exampleshttp://barleyworld.org/css430_09/lecture%208-09/figure-11-04.JPG
    • 35. Naming Restriction EnzymesRestriction enzymes are named according to theorganism from which it was identified.Example: EcoRIE - Escherichiaco - coliR - strain RY13I - 1st enzyme in this strain
    • 36. Practice: Naming RestrictionEnzymesBacillus amyloliquefaciens, strain H, 5thendonuclease identifiedNocardia otitidis, 3rd endonuclease identifiedBamHVNotIII
    • 37. Animation: Restriction Enzymehttp://highered.mcgraw-hill.com/olc/dl/120078/bio37.swfTutorial:http://www.dnalc.org/resources/animations/restriction.html
    • 38. Application of restriction enzymesAny DNA cut with the same restriction can beligated together because they have the samesticky ends that are complementary
    • 39. http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg
    • 40. Recombinant DNA DNA in which genes from two different sources (oftendifferent species) are combined into one molecule Usually the gene of interest is inserted into a bacterialplasmid Why do you think a bacterial plasmid is used?
    • 41. Fig. 20.3Forming Recombinant DNA Restriction enzyme digestion of plasmid and gene ofinterest Hybridization of matching sticky ends on gene ofinterest and plasmid DNA ligase seals gene of interest with plasmid
    • 42. Activity: Recombinant DNAYou are given either a gene of interest (linear) or aplasmid (circular)Cut out your DNADigest it with the given restriction enzymeFind the person with a matching sticky end toform the recombinantAdditional: Research the data pair. Prepare ashort (3-4 sentence) write-up that relates to thetwo terms.
    • 43. Gel ElectrophoresisSize separation of nucleic acids by moving themthrough a gel medium using electric currentCan also be used for protein
    • 44. Gel ElectrophoresisDNA is negatively charge (phosphates) thus willmove toward the positive electrode
    • 45. Making the gelLiquid solutions of thegel medium is pouredinto a mould andallowed to set andsolidifyNotice the comb isplaced at one end of thegel. When it is removedit creates the wellswhere DNA is loaded.
    • 46. Positive (+)electrodeNegative (-)electrodeWhere DNAis loadedonto gelBlue liquidis bufferhttp://cg.scs.carleton.ca/~morin/teaching/compbio/electro.htmlHorizontal Gel
    • 47. Vertical Gelhttp://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg
    • 48. Vertical Gel
    • 49. Types of Gel MediumMedium Agarose PolyacrylamideSource Seaweed extract Artificial polymerResolvingpowerLower resolution Higher resolutionSeparation Nucleic acid Nucleic acid Protein
    • 50. Separation by SizeGel medium is semi-solid and provides resistancefor DNA movement.The more concentrated the gel, the higher theresolving power of the gel.
    • 51. Separation by SizeShort DNAmoves through gel easilytravels furtherLong DNAmoves through gel slower because its size getscaught in the web of polymers that make up a geldoes not move as far
    • 52. http://cg.scs.carleton.ca/~morin/teaching/compbio/f_s02gelelect.gif
    • 53. Other MaterialsLiquid buffer containing ions:Provides a medium for the flow of electric currentPrevents gel from overheating and drying out.Coloured dyes mixed with DNA:track distance travelled on the gel by DNAIncrease density of DNA so that it sinks to bottom ofwells
    • 54. negativeelectrodepositiveelectrodeagarose gelLoading dye
    • 55. Loading dye
    • 56. Animations: gel electrophoresishttp://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
    • 57. DNA Visualization from GelLoading dye doesn’t make DNA on the gel visibleNeed to stain DNA to see it
    • 58. DNA staining: Ethidium bromideA chemical that binds to the DNAcarcinogenic!!!Glows under UV lightUse UV light box to see fluorescent DNA bands
    • 59. DNA stained with ethidum bromideexposed under UV light
    • 60. Nerdy gifts