Honors ~ DNA 1213

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  • DNA-based Technologies Lecture - Dr. Seth Bordenstein 4 different prophage regions 3.2% of the total genome Low GC content similar to host chromosome dsDNA virus Icosohedral head 20.5Kb genome
  • DNA-based Technologies Lecture - Dr. Seth Bordenstein
  • DNA-based Technologies Lecture - Dr. Seth Bordenstein
  • DNA-based Technologies Lecture - Dr. Seth Bordenstein
  • Honors ~ DNA 1213

    1. 1. Molecular BiologyHonors BiologyEdgar
    2. 2. DNA Replication
    3. 3. Exonuclease
    4. 4. Fig. 16-UN5
    5. 5. Fig. 16-13TopoisomeraseHelicasePrimaseSingle-strand bindingproteinsRNAprimer5′5′5′ 3′3′3′
    6. 6. Fig. 16-16b6Templatestrand5′5′3′3′RNA primer 3′5′5′3′113′3′5′5′Okazakifragment123′3′5′5′123′3′5′5′125′5′3′3′Overall direction of replication
    7. 7. Fig. 16-16aOverviewOrigin of replicationLeading strandLeading strandLagging strandLagging strandOverall directionsof replication12
    8. 8. Helicase
    9. 9. Topoisomerase and Helicase
    10. 10. Fig. 20-3-1Restriction siteDNASticky endRestriction enzymecuts sugar-phosphatebackbones.5′3′3′5′1
    11. 11. Fig. 20-3-2Restriction siteDNASticky endRestriction enzymecuts sugar-phosphatebackbones.5′3′3′5′1DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.2One possible combination
    12. 12. Fig. 20-3-3Restriction siteDNASticky endRestriction enzymecuts sugar-phosphatebackbones.5′3′3′5′1One possible combinationRecombinant DNA moleculeDNA ligaseseals strands.3DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.2
    13. 13. Fig. 20-9aMixture ofDNA mol-ecules ofdifferentsizesPowersourceLongermoleculesShortermoleculesGelAnodeCathodeTECHNIQUE12Powersource– ++–
    14. 14. Fig. 20-9bRESULTS
    15. 15. Fig. 20-10NormalalleleSickle-cellalleleLargefragment(b) Electrophoresis of restriction fragmentsfrom normal and sickle-cell alleles201 bp175 bp376 bp(a) DdeI restriction sites in normal andsickle-cell alleles of β-globin geneNormal β-globin alleleSickle-cell mutant β-globin alleleDdeILarge fragmentLarge fragment376 bp201 bp175 bpDdeIDdeIDdeI DdeI DdeI DdeI
    16. 16. Restriction Enzyme Lab• HINTS:• pMAP is 5615bp• There are– 2 PstI sites.– 1 HpaI site.– 1 SspI site• Lambda DNA/PstI:• You should not beable to see beyondthe 805bp band.• Fine the 11,490bpand the 805bp asreference.
    17. 17. Transcription and Translation
    18. 18. To studying theTo studying the Wolbachia within?Wolbachia within?Credit: Mark Taylor
    19. 19. 16S rRNA (ribosomal RNA)16S rRNA (ribosomal RNA) Small ribosomal subunit involved in mRNA translation processSmall ribosomal subunit involved in mRNA translation process Ancient molecule, conserved function, universally distributedAncient molecule, conserved function, universally distributed Helps identify unknown bacterium to genus or species levelsHelps identify unknown bacterium to genus or species levels Present in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cellsPresent in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cells Plays a catalytic and structural role in the ribosomePlays a catalytic and structural role in the ribosome
    20. 20. DNA Barcoding
    21. 21. Cytochrome Oxidase (COI)
    22. 22. Bacteria 16S rRNA and EukaryoticBacteria 16S rRNA and Eukaryotic18S rRNA – Similar!18S rRNA – Similar!
    23. 23. 16S rRNA conservation (red)16S rRNA conservation (red)
    24. 24. Gene Regulation
    25. 25. Fig. 18-6DNASignalGeneNUCLEUSChromatinmodificationChromatinGene availablefor transcriptionExonIntronTailRNACapRNA processingPrimary transcriptmRNA in nucleusTransport to cytoplasmmRNA in cytoplasmTranslationCYTOPLASMDegradationof mRNAProtein processingPolypeptideActive proteinCellular functionTransport to cellulardestinationDegradationof proteinTranscription
    26. 26. Gene Regulation Example 1Activators, Enhancers andTranscription Factors
    27. 27. Fig. 18-8-1Enhancer(distal control elements)Proximalcontrol elementsPoly-A signalsequenceTerminationregionDownstreamPromoterUpstreamDNAExonExon ExonIntron Intron
    28. 28. Fig. 18-8-2Enhancer(distal control elements)Proximalcontrol elementsPoly-A signalsequenceTerminationregionDownstreamPromoterUpstreamDNAExon Exon ExonIntronIntronCleaved 3′ endof primarytranscriptPrimary RNAtranscriptPoly-AsignalTranscription5′ExonExon ExonIntron Intron
    29. 29. Fig. 18-8-3Enhancer(distal control elements)Proximalcontrol elementsPoly-A signalsequenceTerminationregionDownstreamPromoterUpstreamDNAExonExon ExonIntron IntronExon Exon ExonIntronIntronCleaved 3′ endof primarytranscriptPrimary RNAtranscriptPoly-AsignalTranscription5′RNA processingIntron RNACoding segmentmRNA5′ Cap 5′ UTRStartcodonStopcodon 3′ UTR Poly-Atail3′
    30. 30. Fig. 18-9-1Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelement
    31. 31. Fig. 18-9-2Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelementGroup ofmediator proteinsDNA-bendingproteinGeneraltranscriptionfactors
    32. 32. Fig. 18-9-3Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelementGroup ofmediator proteinsDNA-bendingproteinGeneraltranscriptionfactorsRNApolymerase IIRNApolymerase IITranscriptioninitiation complex RNA synthesis
    33. 33. Fig. 18-10ControlelementsEnhancerAvailableactivatorsAlbumin gene(b) Lens cellCrystallin geneexpressedAvailableactivatorsLENS CELLNUCLEUSLIVER CELLNUCLEUSCrystallin genePromoter(a) Liver cellCrystallin genenot expressedAlbumin geneexpressedAlbumin genenot expressed
    34. 34. Gene Regulation Example 2The Operon
    35. 35. Fig. 18-2Regulationof geneexpressiontrpE genetrpD genetrpC genetrpB genetrpA gene(b) Regulation of enzymeproduction(a) Regulation of enzymeactivityEnzyme 1Enzyme 2Enzyme 3TryptophanPrecursorFeedbackinhibition
    36. 36. Fig. 18-3aPolypeptide subunits that make upenzymes for tryptophan synthesis(a) Tryptophan absent, repressor inactive, operon onDNAmRNA 5′Protein InactiverepressorRNApolymeraseRegulatorygenePromoter Promotertrp operonGenes of operonOperatorStop codonStart codonmRNAtrpA5′3′trpR trpE trpD trpC trpBABCDE
    37. 37. Fig. 18-3b-1(b) Tryptophan present, repressor active, operon offTryptophan(corepressor)No RNA madeActiverepressormRNAProteinDNA
    38. 38. Fig. 18-3b-2(b) Tryptophan present, repressor active, operon offTryptophan(corepressor)No RNA madeActiverepressormRNAProteinDNA
    39. 39. Fig. 18-4a(a) Lactose absent, repressor active, operon offDNAProteinActiverepressorRNApolymeraseRegulatorygenePromoterOperatormRNA5′3′NoRNAmadelacI lacZ
    40. 40. Fig. 18-4b(b) Lactose present, repressor inactive, operon onmRNAProteinDNAmRNA 5′InactiverepressorAllolactose(inducer)5′3′RNApolymerasePermease Transacetylaselac operonβ-GalactosidaselacYlacZ lacAlacI
    41. 41. Fig. 18-5(b) Lactose present, glucose present (cAMP levellow): little lac mRNA synthesizedcAMPDNAInactive lacrepressorAllolactoseInactiveCAPlacICAP-binding sitePromoterActiveCAPOperatorlacZRNApolymerasebinds andtranscribesInactive lacrepressorlacZOperatorPromoterDNACAP-binding sitelacIRNApolymerase lesslikely to bindInactiveCAP(a) Lactose present, glucose scarce (cAMP levelhigh): abundant lac mRNA synthesized
    42. 42. Gene Regulation Example 3Epigenetics
    43. 43. Epigenetics
    44. 44. Epigenetics Introhttp://learn.genetics.utah.edu/content/epigenetics/intro/
    45. 45. Utah Epigeneticshttp://learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome
    46. 46. Gene Regulation Example 4RNAi
    47. 47. RNAi
    48. 48. RNAInducedSilencingComplex
    49. 49. Vascular Endothelial Growth Factor
    50. 50. Transformation – RecombinantOrganisms
    51. 51. Cloning Technologies
    52. 52. Fig. 20-4-1Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUE
    53. 53. Fig. 20-4-2Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUERecombinant plasmidsNonrecombinantplasmid
    54. 54. Fig. 20-4-3Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUERecombinant plasmidsNonrecombinantplasmidBacteria carryingplasmids
    55. 55. Fig. 20-4-4Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUERecombinant plasmidsNonrecombinantplasmidBacteria carryingplasmidsRESULTSColony carrying non-recombinant plasmidwith intact lacZ geneOne of manybacterialclonesColony carrying recombinantplasmid with disrupted lacZ gene
    56. 56. mtDNATheories, Molecular Basisand Real-World Application
    57. 57. “The Other Genome”mtDNA
    58. 58. Endosymbiotic Theory
    59. 59. DNA Laboratory atMilton Academy• Isolate DNAfrom cheek cells.• PolymeraseChair Reaction• Electrophoresis• Sequence DNA
    60. 60. mtDNA Control Region
    61. 61. Polymerase Chain Reaction
    62. 62. PCRhttp://www.dnalc.org/resources/spotlight/index.html
    63. 63. Taq DNA Polymerase
    64. 64. Fig. 20-8a5′Genomic DNATECHNIQUETargetsequence3′3′ 5′
    65. 65. Fig. 20-8bCycle 1yields2moleculesDenaturationAnnealingExtensionPrimersNewnucleo-tides3′ 5′325′ 3′1
    66. 66. Fig. 20-8cCycle 2yields4molecules
    67. 67. Fig. 20-8dCycle 3yields 8molecules;2 molecules(in whiteboxes)match targetsequence
    68. 68. http://www.youtube.com/watch?v=CQEaX3MiDowhttp://www.youtube.com/watch?v=x5yPkxCLads&feature=related
    69. 69. Gel Electrophoresis
    70. 70. DNA Sequencing
    71. 71. Chain Termination MethodsSanger Methods
    72. 72. Dye-terminator sequencing
    73. 73. Fig. 20-12DNA(template strand)TECHNIQUERESULTSDNA (templatestrand)DNApolymerasePrimer DeoxyribonucleotidesShortestDideoxyribonucleotides(fluorescently tagged)Labeled strandsLongestShortest labeled strandLongest labeled strandLaserDirectionof movementof strandsDetectorLast baseof longestlabeledstrandLast baseof shortestlabeledstranddATPdCTPdTTPdGTPddATPddCTPddTTPddGTP
    74. 74. Fig. 20-12aDNA(template strand)TECHNIQUEDNApolymerasePrimer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)dATPdCTPdTTPdGTPddATPddCTPddTTPddGTP
    75. 75. Fig. 20-12bTECHNIQUERESULTSDNA (templatestrand)ShortestLabeled strandsLongestShortest labeled strandLongest labeled strandLaserDirectionof movementof strandsDetectorLast baseof longestlabeledstrandLast baseof shortestlabeledstrand
    76. 76. Trace File
    77. 77. Amplification and clonalselection
    78. 78. Kate BatorConnor Johnson
    79. 79. High-throughput sequencingNext-Gen Sequencing
    80. 80. mtDNA Sequencehttp://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html

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