《分子生物学》2-sectionggenemanipula

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,Isolationandmanipulationoffragmentsofanorganismsgenome,Molecularanalysisofproteinsorotherinterestedgeneproducts,Impossiblebydirectpurification,DNAcloning,Impossiblebydirectisolation,Crucial!,Makeallpossible!,Genemanipulation,molecularcloning,geneticengineering,ApplicationsofDNAcloning,Sequencing,hencetoderiveproteinsequence;genomicsIsolationandanalysisofgenepromoteretcInvestigationofprotein/enzyme/RNAfunctioninvariousforms.Identificationmutations,geneticdiseasesBiotechnology:proteinsofpharmaceuticalimportanceTransgenicplantsandanimalsGenetherapy,DNACloning,TechniqueskanRforkanamycin.,back,Earlierplasmiddeveloped,Versatilecloningplasmid,Phagemid(噬菌粒）,Hostsandvectors,Hostorganism/cell:wheretheplasmidsgetmultipliedandpropagatedfaithfully,whichiscrucialforDNAcloning.HostsforDNAcloningvectorProkaryotichost:E.coli(mostcases)Eukaryotichost:YeastSaccharomycescerevisiae(largefragmentsofhumangenome),GeneralfeaturesofaVectorautonomouslyreplicatingDNAindependentofhostsgenome.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,from2to200kbinsize,whichexistinmultiplecopieswithinthehostcells.containanoriginofreplicationandreplicateindependentlyUsuallycarryafewgenes,oneofwhichmayconferresistancetoantibacterialsubstance.Example:amprgeneencodingtheenzymeb-lactamsewhichdegradespenicillinantibioticssuchasampicillin.,PlasmidminipreparationfromE.coli,Plasmids2-20kbinlengththatismuchsmallerthanE.colichromosomalDNA(4600kb),andindependentlysupercoiledResistanttoshearingforceandchemicaldenaturation,thuscanbeisolatedfromthechromosomalDNAeasilysuchasbyalkalinelysis.Minipreparation(miniprep)IsolationofplasmidDNAfromafewmililiters(ml)ofbacterialculture.,Minipreps,GrowthofthecellscontainingplasmidsCollectthecellsbycentrifugationAlkalinelysisresuspensionalkalinelysisneutralizationPhenolextractiontogetridoftheproteincontaminantsEthanolprecipitationtoconcentratethenucleicacidsremained(0.3MNaAc,2-3volethanol).Resuspendinsuitablebuffer:TE10/1,pH8.0(PleasenotethatRNaseAisverybadforthelabworkingwithRNA),AlkalinelysisResuspendthecellsinabuffersolutionLysozymetodigestthecellwall(optional)CelllysisinlysisbuffercontainingSDS(disruptscellmembraneanddenaturesproteins)andNaOH(denaturesDNA)NeutralizationbuffercontainingKOAc(pH5):renaturationofplasmidDNA(supercoiled)andprecipitationofdenaturedproteinsandchromosomalDNA.Centrifugationplasmidinsupernatant(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,thecellsshallbeputat42Cfor1-2mininordertoincreasethetransformationefficiency,Selection