《分子生物学》2-section g gene manipula.ppt

DNACloningDNAcloning DNA克隆Molecularcloning 分子克隆Genemanipulation 基因操作RecombinantDNAtechnology 重组DNA技术Geneticengineering遗传工程 基因工程Biotechnology 生物技术 Geneengineering Enzymeengineering Cell Fermentationengineering Biomedicine Agrobiotechnolgyetc DNAcloning Genemanipulation Toplacearelativelyshortfragmentofagenome whichmightcontainthegeneorothersequenceofinterest inanautonomouslyreplicatingpieceofDNA knownasavector 载体 formingrecombinantDNA whichcanbereplicatedindependentlyoftheoriginalgenome andnormallyinotherhostspeciesaltogether PropagationofthehostorganismcontainingtherecombinantDNAformsasetofgeneticallyidenticalorganism oraclone ThisprocessiscalledDNAcloning BasicprocedureofDNAcloning Vector Genomicfragment restriction PCR cDNA insert Plasmidpreparation vector Restrictiondigestion trimmingtheDNAends Ligation jointheinsertandthevector Transformation introducetheplasmidsintohostcells Analysisoftherecombinants Electrophoresis checkyourDNA DNACloning asimplifiedflowchart Isolationandmanipulationoffragmentsofanorganism sgenome Molecularanalysisofproteinsorotherinterestedgeneproducts Impossiblebydirectpurification DNAcloning Impossiblebydirectisolation Crucial Makeallpossible Genemanipulation molecularcloning geneticengineering ApplicationsofDNAcloning Sequencing hencetoderiveproteinsequence genomicsIsolationandanalysisofgenepromoteretcInvestigationofprotein enzyme RNAfunctioninvariousforms Identificationmutations geneticdiseasesBiotechnology proteinsofpharmaceuticalimportanceTransgenicplantsandanimalsGenetherapy DNACloning Techniques Applications SectionG Genemanipulation DNAcloning subcloning basictechniques sectionH CloningvectorsSectionI Genelibraries screeningSectionJ Analysis usesofclonedDNA Themostbasicconceptsandtechnicaltoolsofmolecularbiology G1DNAcloning anoverview basicconcepts G2PreparationofplasmidDNAG3Ligation transformationandanalysisofrecombinants SectionGGenemanipulation G1DNAcloning anoverview HostsandvectorsSubcloningDNAlibrariesScreeninglibrariesAnalysisofaclone back Plasmidasvectors Plasmids 质粒 small extrachromosomalcircularmolecules from2to 200kbinsize whichexistinmultiplecopieswithinthehostcells containanoriginofreplicationandreplicateindependentlyUsuallycarryafewgenes oneofwhichmayconferresistancetoantibacterialsubstance Example ampRgeneencodingtheenzymeb lactamasewhichdegradespenicillinantibioticssuchasampicillin kanRforkanamycin back Earlierplasmiddeveloped Versatilecloningplasmid Phagemid 噬菌粒 Hostsandvectors Hostorganism cell wheretheplasmidsgetmultipliedandpropagatedfaithfully whichiscrucialforDNAcloning HostsforDNAcloningvectorProkaryotichost E coli mostcases Eukaryotichost YeastSaccharomycescerevisiae largefragmentsofhumangenome GeneralfeaturesofaVectorautonomouslyreplicatingDNAindependentofhost sgenome EasilytobeisolatedfromthehostcellMostarecircular somearelinearContainsatleastoneselectivemarker whichallowshostcellscontainingthevectortobeselectedamongstthosewhichdonot Containsamultiplecloningsite MCS Typesofvectors CloningvectorsExpressionvectorsIntegrationvectorsViralvectors Cloningvectors allowingtheexogenousDNAtobeinserted stored andmanipulatedatDNAlevel E colicloningvector plasmids bacteriophages landM13 plasmid bacteriophagelhybrids cosmids Yeastcloningvector yeastartificialchromosomes YACs Expressionvectors allowingtheexogenousDNAtobeinsertedandexpressed PromoterandterminatorforRNAtranscriptionarerequired bacterialexpressionvectorsyeastexpressionvectorsmammalianexpressionvectors Integrationvectors allowingtheexogenousDNAtobeinsertedandintegratedintoachromosomalDNAafteratransformation Theintegrationiseitherrandominsertionorconductedbyhomologousrecombinationbetweenthehomologoussequencesharedbytheplasmidandthegenomeoftherecipientcells bacterialintegrationvectors AgrobacteriumtumefaciensTiplasmidisusedtointegrateDNAintoplantgenome yeastintegrationvectorsMammalianintegrationvector genetargeting back Viralvectors Bacterialphage Lambda M13Insect baculovirusesMammalianviruses SV40 poxvirus adenovirus retrovirusesPlantviruses TMV PVX back Adenoviralvectorsystem Plantvirusvector PVX potatovirusX SubcloningTransferofafragmentofclonedDNAfromonevectortoanother Enablesustoinvestigateashortregionofalargeclonedfragmentinmoredetail Totransferagenefromoneplasmidtoavectordesignedtoexpressitinaparticularspecies PreparationofplasmidscontainingaclonedDNAfragment insert Plasmidpreparation vector Restrictiondigestion trimmingtheDNAends Separation purification ligation jointheinsertandthevector Transformation selectionoftransformants introducetheplasmidsintohostcells Analysisoftherecombinants DNASubcloning aflowchart Restrictionendonuclease back AgroseGelElectrophoresis checkyourDNAateachstepSeparationandPurificationofDNAfragmentsofinterestsAnalysisofrecombinantplasmids ladder Restrictionanalysisofaplasmid DNAlibraries DNAlibrariesaresetsofDNAclones eachofwhichhasbeenderivedfromtheinsertionofadifferentfragmentintoavectorfollowedbypropagationinthehost AcloneisageneticallydistinctindividualorsetofidenticalindividualsGenomiclibrariescDNAlibraries GenomiclibrariespreparedformrandomfragmentsofgenomicDNA whichmaybeinefficienttofindagenebecauseofthehugeabundanceofthenon codingDNA cDNAlibrariesDNAcopies cDNA synthesizedfromthemRNAbyreversetranscriptionareinsertedintoavectortoformacDNAlibrary Muchmoreefficientinidentifyingagene butdonotcontainDNAcodingforfunctionalRNAornoncodingsequence Screeninglibraries Colonyorplaquehybridization RadiolabeledprobescomplementarytoaregionoftheinterestedgeneProbes AnoligonucleotidederivedfromthesequenceofaproteinproductofthegeneADNAfragment oligofromarelatedgeneofanotherspeciesPCRproductPlatingthecellscarryingthelibraryColonyorplaqueliftonmembraneandthenhybridizewiththelabeledprobe SearchingthegenesofinterestinaDNAlibrary Screeninglibraries Expressionscreening SpecificantibodytogeneproductScreeninglibrarybyPCR SpeciallypreparedlibraryFunctionalscreening Complementationtoalethalphenotype possibleforotherkindsofpositivescreening SearchingthegenesofinterestinaDNAlibrary IdentifytheproteinproductofaninterestedgeneProteinactivityWesternblottingusingaspecificantibodyInvivoexpressionandfunctionalassay back Analysisofaclone Restrictionmapping digestionofthewithrestrictionenzymes SequencingtheclonedDNA back YoumayhavetofullyunderstandthefunctionandapplicationofalltheenzymeslistedinTable1ifyouwanttomanipulategenes EnzymescommonlyusedinDNAcloning AlkalinephosphotaseReversetranscriptase DNAligase T4 DNApolI Klenowfragment T4 TaqExunucleaseIIIMungbeannucleaseandS1nucleasePolynucleotidekinaseRestrictionenzymes e g EcoRI HindIIIRNaseA RNaseHT7 T3andSP6RNApolymerasesTerminaltransferase back G2 PreparationofplasmidDNA 1 PlasmidasvectorsPlasmidminipreparationAlkalinelysisPhenolextractionEthanolprecipitationCesiumchloridegradientpurification Plasmidasvectors Plasmids small extrachromosomalcircularmolecules from2to 200kbinsize whichexistinmultiplecopieswithinthehostcells containanoriginofreplicationandreplicateindependentlyUsuallycarryafewgenes oneofwhichmayconferresistancetoantibacterialsubstance Example amprgeneencodingtheenzymeb lactamsewhichdegradespenicillinantibioticssuchasampicillin PlasmidminipreparationfromE coli Plasmids 2 20kbinlengththatismuchsmallerthanE colichromosomalDNA 4600kb andindependentlysupercoiledResistanttoshearingforceandchemicaldenaturation thuscanbeisolatedfromthechromosomalDNAeasilysuchasbyalkalinelysis Minipreparation miniprep IsolationofplasmidDNAfromafewmililiters ml ofbacterialculture Minipreps GrowthofthecellscontainingplasmidsCollectthecellsbycentrifugationAlkalinelysisresuspension alkalinelysis neutralizationPhenolextractiontogetridoftheproteincontaminantsEthanolprecipitationtoconcentratethenucleicacidsremained 0 3MNaAc 2 3volethanol Resuspendinsuitablebuffer TE10 1 pH8 0 PleasenotethatRNaseAisverybadforthelabworkingwithRNA AlkalinelysisResuspendthecellsinabuffersolutionLysozymetodigestthecellwall optional CelllysisinlysisbuffercontainingSDS disruptscellmembraneanddenaturesproteins andNaOH denaturesDNA NeutralizationbuffercontainingKOAc pH5 renaturationofplasmidDNA supercoiled andprecipitationofdenaturedproteinsandchromosomalDNA Centrifugation plasmidinsupernatant lysate Growthecell Harvestthecellbycentrifugation Alkalinelysisofthecell Resuspendthecellpellet neutralization Phenolextraction Ethanolprecipitation CsClgradientpurification PurificationofplasmidDNAbyCesiumchloridegradientcentrifugation CsClgradientpurificationisthelaststepoflargescaleplasmidDNApurificationLaboriousBestfortheproductionofverypuresupercoiledplasmidDNAThepresenceofethidiumbromide EB isimportant BindingofEBtoDNAwillunwindtheDNAandreducetheDNAdensitySupercoildedDNAbindlessEBthanlinearDNAornickedDNA thushasahigherdensity SupercoiledDNAmaybepurifiedfromprotein RNAchromosomalDNAandnickedplasmidDNAinonestep back Agarosegelelectrophoresis supercoiled nicked IsolationoffragmentsandAgarosegelelectrophoresis insert RestrictiondigestionAgarosegelelectrophoresis 3 GelexcisionandpurificationLigationwithvectorTransformation back G4 Ligation transformationandanalysisofrecombinants AlkalinephosphataseDNAligation recombinantDNAmoleculesTransformation selectionTransformationefficiency4 Screeningtransformants5 GrowthandstorageoftransformantsGelanalysisFragmentorientation AlkalinephosphataseSinglerestrictionenzymedirectedcloningRemovesthephosphategroupsfromthe5 endsofthevectorDNAlinearizedbyasinglerestrictionenzymetopreventtheself ligationofthevectorDNAuponthefollowedligation DNAligationCovalentlyjointheDNAmoleculeswiththebase pairingcohesiveends orbluntends ifthe5 endshavephosphategroups Xifthevectorisphosphorylated RecombinantDNAmolecules Theuseofalkalinephosphatasetopreventreligationofvectormolecules G OHCTTAA OH Transformationandselection Competentcells 感受态细胞 E colicellstreatedwithCa2 solutionaresusceptibletotakeupexogenousDNA Enzymesinvolvedinhostcelldefending suchasrestriction modificationsystemaresuppressed Transformation 转化 aprocessofuptakeofexogenousDNAbycompetentcells Heat shock 热休克 AftertheDNAisuptaken thece