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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 Honors ~ DNA 1213 Presentation Transcript

  • Molecular BiologyHonors BiologyEdgar
  • DNA Replication
  • Exonuclease
  • Fig. 16-UN5
  • Fig. 16-13TopoisomeraseHelicasePrimaseSingle-strand bindingproteinsRNAprimer5′5′5′ 3′3′3′
  • 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
  • Fig. 16-16aOverviewOrigin of replicationLeading strandLeading strandLagging strandLagging strandOverall directionsof replication12
  • Helicase
  • Topoisomerase and Helicase
  • Fig. 20-3-1Restriction siteDNASticky endRestriction enzymecuts sugar-phosphatebackbones.5′3′3′5′1
  • 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
  • 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
  • Fig. 20-9aMixture ofDNA mol-ecules ofdifferentsizesPowersourceLongermoleculesShortermoleculesGelAnodeCathodeTECHNIQUE12Powersource– ++–
  • Fig. 20-9bRESULTS
  • 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
  • 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.
  • Transcription and Translation
  • To studying theTo studying the Wolbachia within?Wolbachia within?Credit: Mark Taylor
  • 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
  • DNA Barcoding
  • Cytochrome Oxidase (COI)
  • Bacteria 16S rRNA and EukaryoticBacteria 16S rRNA and Eukaryotic18S rRNA – Similar!18S rRNA – Similar!
  • 16S rRNA conservation (red)16S rRNA conservation (red)
  • Gene Regulation
  • Fig. 18-6DNASignalGeneNUCLEUSChromatinmodificationChromatinGene availablefor transcriptionExonIntronTailRNACapRNA processingPrimary transcriptmRNA in nucleusTransport to cytoplasmmRNA in cytoplasmTranslationCYTOPLASMDegradationof mRNAProtein processingPolypeptideActive proteinCellular functionTransport to cellulardestinationDegradationof proteinTranscription
  • Gene Regulation Example 1Activators, Enhancers andTranscription Factors
  • Fig. 18-8-1Enhancer(distal control elements)Proximalcontrol elementsPoly-A signalsequenceTerminationregionDownstreamPromoterUpstreamDNAExonExon ExonIntron Intron
  • Fig. 18-8-2Enhancer(distal control elements)Proximalcontrol elementsPoly-A signalsequenceTerminationregionDownstreamPromoterUpstreamDNAExon Exon ExonIntronIntronCleaved 3′ endof primarytranscriptPrimary RNAtranscriptPoly-AsignalTranscription5′ExonExon ExonIntron Intron
  • 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′
  • Fig. 18-9-1Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelement
  • Fig. 18-9-2Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelementGroup ofmediator proteinsDNA-bendingproteinGeneraltranscriptionfactors
  • Fig. 18-9-3Enhancer TATAboxPromoterActivatorsDNAGeneDistal controlelementGroup ofmediator proteinsDNA-bendingproteinGeneraltranscriptionfactorsRNApolymerase IIRNApolymerase IITranscriptioninitiation complex RNA synthesis
  • Fig. 18-10ControlelementsEnhancerAvailableactivatorsAlbumin gene(b) Lens cellCrystallin geneexpressedAvailableactivatorsLENS CELLNUCLEUSLIVER CELLNUCLEUSCrystallin genePromoter(a) Liver cellCrystallin genenot expressedAlbumin geneexpressedAlbumin genenot expressed
  • Gene Regulation Example 2The Operon
  • Fig. 18-2Regulationof geneexpressiontrpE genetrpD genetrpC genetrpB genetrpA gene(b) Regulation of enzymeproduction(a) Regulation of enzymeactivityEnzyme 1Enzyme 2Enzyme 3TryptophanPrecursorFeedbackinhibition
  • 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
  • Fig. 18-3b-1(b) Tryptophan present, repressor active, operon offTryptophan(corepressor)No RNA madeActiverepressormRNAProteinDNA
  • Fig. 18-3b-2(b) Tryptophan present, repressor active, operon offTryptophan(corepressor)No RNA madeActiverepressormRNAProteinDNA
  • Fig. 18-4a(a) Lactose absent, repressor active, operon offDNAProteinActiverepressorRNApolymeraseRegulatorygenePromoterOperatormRNA5′3′NoRNAmadelacI lacZ
  • Fig. 18-4b(b) Lactose present, repressor inactive, operon onmRNAProteinDNAmRNA 5′InactiverepressorAllolactose(inducer)5′3′RNApolymerasePermease Transacetylaselac operonβ-GalactosidaselacYlacZ lacAlacI
  • 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
  • Gene Regulation Example 3Epigenetics
  • Epigenetics
  • Epigenetics Introhttp://learn.genetics.utah.edu/content/epigenetics/intro/
  • Utah Epigeneticshttp://learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome
  • Gene Regulation Example 4RNAi
  • RNAi
  • RNAInducedSilencingComplex
  • Vascular Endothelial Growth Factor
  • Transformation – RecombinantOrganisms
  • Cloning Technologies
  • Fig. 20-4-1Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUE
  • Fig. 20-4-2Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUERecombinant plasmidsNonrecombinantplasmid
  • Fig. 20-4-3Bacterial cellBacterialplasmidlacZ geneHummingbirdcellGene of interestHummingbirdDNA fragmentsRestrictionsiteStickyendsampRgeneTECHNIQUERecombinant plasmidsNonrecombinantplasmidBacteria carryingplasmids
  • 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
  • mtDNATheories, Molecular Basisand Real-World Application
  • “The Other Genome”mtDNA
  • Endosymbiotic Theory
  • DNA Laboratory atMilton Academy• Isolate DNAfrom cheek cells.• PolymeraseChair Reaction• Electrophoresis• Sequence DNA
  • mtDNA Control Region
  • Polymerase Chain Reaction
  • PCRhttp://www.dnalc.org/resources/spotlight/index.html
  • Taq DNA Polymerase
  • Fig. 20-8a5′Genomic DNATECHNIQUETargetsequence3′3′ 5′
  • Fig. 20-8bCycle 1yields2moleculesDenaturationAnnealingExtensionPrimersNewnucleo-tides3′ 5′325′ 3′1
  • Fig. 20-8cCycle 2yields4molecules
  • Fig. 20-8dCycle 3yields 8molecules;2 molecules(in whiteboxes)match targetsequence
  • http://www.youtube.com/watch?v=CQEaX3MiDowhttp://www.youtube.com/watch?v=x5yPkxCLads&feature=related
  • Gel Electrophoresis
  • DNA Sequencing
  • Chain Termination MethodsSanger Methods
  • Dye-terminator sequencing
  • Fig. 20-12DNA(template strand)TECHNIQUERESULTSDNA (templatestrand)DNApolymerasePrimer DeoxyribonucleotidesShortestDideoxyribonucleotides(fluorescently tagged)Labeled strandsLongestShortest labeled strandLongest labeled strandLaserDirectionof movementof strandsDetectorLast baseof longestlabeledstrandLast baseof shortestlabeledstranddATPdCTPdTTPdGTPddATPddCTPddTTPddGTP
  • Fig. 20-12aDNA(template strand)TECHNIQUEDNApolymerasePrimer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)dATPdCTPdTTPdGTPddATPddCTPddTTPddGTP
  • Fig. 20-12bTECHNIQUERESULTSDNA (templatestrand)ShortestLabeled strandsLongestShortest labeled strandLongest labeled strandLaserDirectionof movementof strandsDetectorLast baseof longestlabeledstrandLast baseof shortestlabeledstrand
  • Trace File
  • Amplification and clonalselection
  • Kate BatorConnor Johnson
  • High-throughput sequencingNext-Gen Sequencing
  • mtDNA Sequencehttp://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html