DNAToolsBiotechnology
Biotechnologymanipulation ofbiological organisms(usually with DNAitself)
Molecular Genetic TechniquesIsolating DNAAmplifying DNAIn vitro: Polymerase Chain reaction – Fig 20.7In vivo: Transfor...
DNA IsolationBefore DNA can bemanipulated, it needs tobe isolated from thecells.
DNA IsolationSteps:1. Disrupt cellmembrane with adetergent2.Precipitate DNAwith ethanol3.Retrieveprecipitated DNAand store...
DNA Amplification In vitro: PCRPolymerase chain reactionPurpose: to increase the amount of DNADNA amplificationCreate ...
BrainstormWhat are the major steps in DNA replication?What substances do you need in each step of DNAreplication?
Comparing DNA ReplicationSteps Natural PCRUnwindingOri on DNA templateHelicaseDNA templateHeatPrimingRNA primerPrima...
PCR MaterialsDNA templateNucleotidesDNAP (Taq polymerase)DNA primers
PCR Cycle1. Denaturation(95°C)2. Primer annealing(50°C - 65°C)3. Synthesis (72°C)http://www.foodsafetywatch.com/public/ima...
PCR Animationhttp://highered.mcgraw-hill.com/olc/dl/120078/micro15.swfhttp://www.dnalc.org/resources/3d/19-polymerase-ch...
PCRAt which cycle do you get thecorrectly 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 polymerization2 primers u...
Primers identify target sequencehttp://www.mun.ca/biology/scarr/PCR_detailed.jpg
What’s up with Taq?Why can’t we use the DNAP that is found in mostbacteria?
Taq PolymeraseDNAP Isolated from bacteria(Thermus aquaticus) that live inhot springsHeat stable enzyme (canwithstand ext...
Water baths to thermal cyclerBefore taq3 water baths at 3temperaturesre-addition of DNAP atthe beginning of eachsynthesi...
PCR EfficiencyThermal cycler:has a plate thatheats and coolsProcess takes 1–2hoursAfter 30 cycles, 230(more than abill...
Kary MullisDeveloped PCR in 1986Received Nobel Prize in 1993Interviews Naming PCRhttp://www.dnalc.org/view/15138-Naming-P...
Procedure and RationaleTutorialshttp://www.sumanasinc.com/webcontent/animations/content/pcr.htmlhttp://www.dnalc.org/dd...
DNA Amplification in vivoBacterial cells are transformed (uptake externalDNA)Bacteria are grown in a liquid mediumAs th...
Methods of BacterialTransformationtreat bacteria to make cell walls permeable touptake genetic materialCold CaCl2 treatm...
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinitionDNAamplificationAdvantage ofsystem
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environ...
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environ...
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environ...
Restriction EnzymesBiological “scissors”Endonuclease: breakphosphodiester bondswithin a nucleotide chain(as opposed to a...
Restriction Enzyme FunctionFound naturally in bacteria“immune system” of bacteriaprotect bacteria against intruding DNA...
Restriction SiteEach restriction enzyme has a specific sequencethat it recognizes and a specific location on thesequence ...
Restriction Enzyme Digestion Enzyme cuts at thesame site on both strands Restriction fragments:pieces of DNA created byr...
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 ...
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...
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 ...
Restriction Enzyme Exampleshttp://barleyworld.org/css430_09/lecture%208-09/figure-11-04.JPG
Naming Restriction EnzymesRestriction enzymes are named according to theorganism from which it was identified.Example: E...
Practice: Naming RestrictionEnzymesBacillus amyloliquefaciens, strain H, 5thendonuclease identifiedNocardia otitidis, 3r...
Animation: Restriction Enzymehttp://highered.mcgraw-hill.com/olc/dl/120078/bio37.swfTutorial:http://www.dnalc.org/resour...
Application of restriction enzymesAny DNA cut with the same restriction can beligated together because they have the same...
http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg
Recombinant DNA DNA in which genes from two different sources (oftendifferent species) are combined into one molecule Us...
Fig. 20.3Forming Recombinant DNA Restriction enzyme digestion of plasmid and gene ofinterest Hybridization of matching s...
Activity: Recombinant DNAYou are given either a gene of interest (linear) or aplasmid (circular)Cut out your DNADigest ...
Gel ElectrophoresisSize separation of nucleic acids by moving themthrough a gel medium using electric currentCan also be...
Gel ElectrophoresisDNA is negatively charge (phosphates) thus willmove toward the positive electrode
Making the gelLiquid solutions of thegel medium is pouredinto a mould andallowed to set andsolidifyNotice the comb ispla...
Positive (+)electrodeNegative (-)electrodeWhere DNAis loadedonto gelBlue liquidis bufferhttp://cg.scs.carleton.ca/~morin/t...
Vertical Gelhttp://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg
Vertical Gel
Types of Gel MediumMedium Agarose PolyacrylamideSource Seaweed extract Artificial polymerResolvingpowerLower resolution Hi...
Separation by SizeGel medium is semi-solid and provides resistancefor DNA movement.The more concentrated the gel, the hi...
Separation by SizeShort DNAmoves through gel easilytravels furtherLong DNAmoves through gel slower because its size g...
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 currentPrevents gel from overhe...
negativeelectrodepositiveelectrodeagarose gelLoading dye
Loading dye
Animations: gel electrophoresishttp://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
DNA Visualization from GelLoading dye doesn’t make DNA on the gel visibleNeed to stain DNA to see it
DNA staining: Ethidium bromideA chemical that binds to the DNAcarcinogenic!!!Glows under UV lightUse UV light box to s...
DNA stained with ethidum bromideexposed under UV light
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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 of "Biotechnology basic techniques"

    1. 1. DNAToolsBiotechnology
    2. 2. Biotechnologymanipulation ofbiological organisms(usually with DNAitself)
    3. 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. 4. DNA IsolationBefore DNA can bemanipulated, it needs tobe isolated from thecells.
    5. 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. 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. 7. BrainstormWhat are the major steps in DNA replication?What substances do you need in each step of DNAreplication?
    8. 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. 9. PCR MaterialsDNA templateNucleotidesDNAP (Taq polymerase)DNA primers
    10. 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. 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. 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. 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. 14. Primers identify target sequencehttp://www.mun.ca/biology/scarr/PCR_detailed.jpg
    15. 15. What’s up with Taq?Why can’t we use the DNAP that is found in mostbacteria?
    16. 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. 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. 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. 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. 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. 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. 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. 23. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinitionDNAamplificationAdvantage ofsystem
    24. 24. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationAdvantage ofsystem
    25. 25. in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationPCR Bacterial cell transformedwith DNAAdvantage ofsystem
    26. 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. 27. Restriction EnzymesBiological “scissors”Endonuclease: breakphosphodiester bondswithin a nucleotide chain(as opposed to at theends of a chain)Fig. 20.2
    28. 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. 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. 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. 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. 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. 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. 34. Restriction Enzyme Exampleshttp://barleyworld.org/css430_09/lecture%208-09/figure-11-04.JPG
    35. 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. 36. Practice: Naming RestrictionEnzymesBacillus amyloliquefaciens, strain H, 5thendonuclease identifiedNocardia otitidis, 3rd endonuclease identifiedBamHVNotIII
    37. 37. Animation: Restriction Enzymehttp://highered.mcgraw-hill.com/olc/dl/120078/bio37.swfTutorial:http://www.dnalc.org/resources/animations/restriction.html
    38. 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. 39. http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg
    40. 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. 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. 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. 43. Gel ElectrophoresisSize separation of nucleic acids by moving themthrough a gel medium using electric currentCan also be used for protein
    44. 44. Gel ElectrophoresisDNA is negatively charge (phosphates) thus willmove toward the positive electrode
    45. 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. 46. Positive (+)electrodeNegative (-)electrodeWhere DNAis loadedonto gelBlue liquidis bufferhttp://cg.scs.carleton.ca/~morin/teaching/compbio/electro.htmlHorizontal Gel
    47. 47. Vertical Gelhttp://www.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg
    48. 48. Vertical Gel
    49. 49. Types of Gel MediumMedium Agarose PolyacrylamideSource Seaweed extract Artificial polymerResolvingpowerLower resolution Higher resolutionSeparation Nucleic acid Nucleic acid Protein
    50. 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. 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. 52. http://cg.scs.carleton.ca/~morin/teaching/compbio/f_s02gelelect.gif
    53. 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. 54. negativeelectrodepositiveelectrodeagarose gelLoading dye
    55. 55. Loading dye
    56. 56. Animations: gel electrophoresishttp://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
    57. 57. DNA Visualization from GelLoading dye doesn’t make DNA on the gel visibleNeed to stain DNA to see it
    58. 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. 59. DNA stained with ethidum bromideexposed under UV light
    60. 60. Nerdy gifts
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