生化2014c21generecombinationandrecombinationtechnology

Chapter14,DNARecombinationandRecombinationDNATechnology,Dept.ofBiochemistryandMolecularBiologyProfessorWuYaosheng2014-06,MainContents,RecombinantDNATechnology,DNArecombination,GeneRecombinationandClinic,3,KeyPoints,DNArecombinationmanner,homologousrecombinationDNAcloning,targetgene,toolenzymes,RE,vectors,plasmid,hostcell,Ecoli.LigationoftargetgeneandvectorMethodanditsmechanismofscreeningpositiveclonesAlphamutualcomplementexperimentGenediagnosisandgenetherapy,4,SectionOne,DNARecombination,5,DNARecombinationManners,6,Therecombinationwhichoccurbetweenthehomologoussequencescalledashomologousrecombination,orgeneralrecombination）。,Forexample,thehollidaymodelinE.coli,1.Homologousrecombination,ItisthebasictypeofDNArecombination,7,FourkeystepsofHollidaymodel:,Twohomologouschromosomesalignmutuallytrimness,FormationofintermediateHollidaybyonestrandbreakofDNAandjoinwithanotherstrandofanotherDNA,FormationofheterogousdoublestrandDNAbymovementofbreakfork,IntermediateHollidaybesplited,andmodified,toformtwodoublestrandrecombinantDNAs：,8,patchrecombinant（onrightsite）：切开的链与原来断裂的是同一条链，重组体含有一段异源双链区，其两侧来自同一亲本DNA。,splicerecombinant（onleftsite）：切开的链并非原来断裂的链，重组体异源双链区的两侧来自不同亲本DNA。,9,Holiday中间体,10,内切酶(ruvC),内切酶(ruvC),DNA连接酶,DNA连接酶,片段重组体,拼接重组体,11,片段重组体（patchrecombinant）,拼接重组体（splicerecombinant）,问题：1、形成哪种重组体对基因重组更有效？是拼接重组体还是片段重组体？2、同源重组原理有何应用？,“geneknockout”,“genetargeting”,12,获07诺贝尔生理或医学奖科学家,“在涉及胚胎干细胞和哺乳动物DNA重组方面的一系列突破性发现”及“基因打靶”,马里奥.卡佩奇奥利弗.史密斯马丁.埃文斯,13,Embryonalstem,14,15,2.Transferandrecombinantofbacteriagene,(1)Conjugation(接合作用),Whenthecell-cellorbacteriacontactwitheachotherbypili,theplasmidDNAfromasinglecell(bacteria)canbetransferredtoanothercell(bacteria).ThisprocessofDNAtransferiscalledconjugation.,16,IspossibletoacceptplasmidsuchasFfactor,CircularsmalldoublestrandDNAseparatedwithbacterialchromosome,Plasmid,17,(2)Transformation(转化作用),ObtainedthroughtheautomaticorartificialsupplyofexogenousDNA,thereceptorcellsorculturedcellswouldacquirenewgeneticphenotype.Thisprocessisknownasthetransformation.,18,Forexample：whenbacteriolysis,thecleavageofDNAfragmentsisuptakenbyanotherbacterial.,19,MultipleChoices,1.TheintegrationbetweentwospecificsitesoftwoDNAmolecularsequencescatalyzedbyintegratedenzymeisknownas:,A.sitespecificrecombinationB.homologousrecombinationC.generalrecombinationD.randomrecombinationE.artificialrecombination,20,MultipleChoices,2.Transformationmeans:,A.phageinfectionB.translocationofageneC.UptakeofforeignDNAtocausethechangeofthetypeofcellbiologyD.toproducepointmutationE.tocauseframeshiftmutation,21,MultipleChoices,3.Therecombinationoccurredbetweenhomologoussequencesiscalled,A.sitespecificrecombinationB.non-sitespecificrecombinationC.generalrecombinationD.randomrecombinationE.artificialrecombination,22,SectionTwo,RecombinantDNATechnology,23,24,25,Thebasicstepsingenecloning,(1)Toget,(2)Toconstruct,(3)Totransport,(4)Toamplify,(5)Tofind,26,(1)SomebasicconceptsaboutDNAcloning,Genecloning,Molecularcloning,RecombinantDNAtechnology,Geneticengineering,27,Thebasicelementsforcloning,1.Targetgenes,2.Toolenzymes,3.Vectors,4.Hostcells,Thebasicrequirements,28,(2)ToolEnzymes,Nucleasescut,shortenordegradenucleicacidmoleculesLigasesjoinnucleicacidmoleculestogetherPolymerasemakecopiesofmoleculesModifyingenzymesremoveoraddchemicalgroupsTopoisomerasesintroduceorremovesupercoilsfromcovalentlyclosed-circularDNA,29,Nucleases,DegradeDNAmoleculesbybreakingthephosphodiesterbonds,ExonucleasesremoveoneofnucleotideresiduesatatimefromtheendofaDNAmoleculeEndonucleasesareabletobreakinternalphosphodiesterbondswithinaDNAmolecule,RestrictionEndonuclease,Therearetwodifferentkindsofnucleases,30,RestrictionEndonucleases,Theinitialobservationthatledtotheeventualdiscoveryofrestrictionendonucleases(RE)wasmadeintheearly1950s,RestrictionoccursbecausethatbacteriumproducesrestrictionendonucleasesthatdegradesthephageDNA,31,ThediscoveryoftheseenzymesledtoNobelprizesforW.Arber,H.SmithandD.Nathansin1978,ThreedifferentclassesofREhavebeenrecognized,butthemostimportantoneisREIIwhichisusedinDNAmanipulation,32,TheNobelPrizeinPhysiologyorMedicine1978,ArberW,NathansD,SmithH,forthediscoveryofrestrictionenzymesandtheirapplicationtoproblemsofmoleculargenetics,33,ThecharactersofRE,Generally,46basesarefound,mostly6bases,afewof810basesThesequencesdiscriminatedusuallyarepalindromestructureTocutthedoublestrandsofDNAatspecialsitesandtoyieldtwokindsofends:bluntendsandstickyends,34,StickyendsandBluntends:,Stickyorcohesiveends:TheresultingDNAfragmentshaveshortsingle-strandedoverhangsateachendBasepairingbetweenthemcansticktheDNAmoleculebacktogetheragainRestrictionendonucleaseswithdifferentrecognitionsequencesmayproducethesamestickyends,eg:BamHI(GGATCC)andBglII(AGATCT),35,5-GGTGAATTCAGC-33-CCACTTAAGTCG5,5-TTGCTGCAGAAG-33-AACGACGTCTTC5,5-stickyend(EcoRI),3-stickyend(PstI),5-GGTGAATTCAGC-33-CCACTTAAGTCG5,+,5-TTGCTGCAGAAG-33-AACGACGTCTTC5,+,36,Bluntendorflushend,Theligationefficiencybetweenthebluntendsisnotashighasthatofthestickyterminus.,SmaI,Makeasimpledouble-strandedcutinthemiddleoftherecognitionsequence,37,NamingofRE,EscherichiacoliRY13I,EcoRI,Thegenusnameofbacteria,Thespeciesnameofbacteria,Thestrainnameofbacteria,TheorderoftheREfoundinbacteria,REsareusuallynamedafterthebacteriumfromwhichtheyareisolated.,38,Ligases,Torepairsingle-strandedbreaksindouble-strandedDNAmoleculesduringDNAreplication,Tojointwoindividualfragmentsofdouble-strandedDNAtogether,39,Ligaseapplication,40,DNAligase,5,3,3,5,5,3,5,3,ATP,ADP,41,Polymerases,SynthesizeanewstrandofDNAcomplementarytoanexistingDNAorRNAtemplate,FourtypesofDNApolymeraseareusedroutinelyingeneticengineering,42,Polymerases,DNApolymeraseI:SynthesizesdsDNAbyformationofa5,3-phosphodiesterbondKlenowfragment:ComefromDNApolymeraseIwithouttheN-terminalfragmentReversetranscriptase:SynthesizesDNAfromRNAtemplateTaqDNApolymerase:UsedinthePCR,comefrombacteriumThermusaquaticus,43,DNAmodifyingenzymes,AlkalinephosphataseFromE.coli,calfintestinaltissueRemovesthephosphategrouppresentatthe5-terminusofaDNAmolecule,PolynucleotidekinaseFromE.coliinfectedwithT4phageHasthereverseeffectofalkalinephosphatase,addingphosphategroupontofree5-terminus,44,Pi,Pi,Pi,Pi,Alkalinephosphatase,Topreventtheplasmidself-cyclization,45,Terminaldeoxynucleotidyltransferasefromcalfthymustissueaddsoneormoredeoxyribonucleotidesontothe3-terminusofaDNA,DNAmodifyingenzymes,Pi,Pi,A,A,A,A,A,A,A,46,KeyPoints,RestrictionEndonuclease,RECharacters,FunctionsandApplication,ToolEnzymes,Nucleases,Ligase,Polymerase,Modifyingenzymes,Topoisomerases,47,cDNA,cDNAlibrary,GenomicDNA,Genomiclibrary,PCRproducts,ArtificialsynthesisDNAfragments,(3)TargetDNA,48,PurificationofDNAfromlivingcells,PreparationoftotalcellDNA(RNA)PreparationofplasmidDNAPreparationofbacteriophageDNA,49,CloningVector,(4)Vectors,ExpressionVector,50,Cloningvector,CloningvectorsareDNAswhichcancarrytargetgenes,transferthemintotherecipientcells.,51,Plasmids,Basiccharactersofplasmids,Small(lessthan10kb),Circular,duplexmoleculesofDNAcontainingmultiplecloningsitesExistatloworhighcopieswithinbacteria,butusefulplasmidpresentinmultiplecopiesContainselectablemarkers,eg:antibioticresistancecapabilityconferredtobacteria,52,Plasmids,Basiccharactersofplasmids,Replicateindependentlyfrombacterialcells,whichpossessatleastanoriginsiteofreplication,Afewtypesofplasmidarealsoabletoreplicatebyinsertingthemselvesintothebacterialchromosome,Multiplywithincellsquiteindependentlyfrombacterialchromosome,53,54,LacZ-galactosidasegene,Multiplecloningsite(MCS),Ampicillinresistancegene,55,Commonusedphages,Bacteriophage,AlineardsDNAapproximately49KbinlengthAfterinfection,itcanformcircularstructuresThephageDNAistransferedintobacterialcells,BacteriophageM13,AcircularssDNA,andhasbeenusedforsequencingofaclonedtargetDNAfragment,56,Cosmids,BacterialArtificialChromosome(BAC)andYeastArtificialChromosome(YAC),Virusareusedasvectors,eg:retro-virus,adeno-virus,adenoassociatedvirus,etc,OtherVectors,57,KeyPoints:Plasmid,Containingmultiplecloningsites,Vectors,Plasmid,Bacteriophage(,M13),Cosmids,BAC,YAC,VirusDNA,Containingselectablemarkers,Replicateindependently,58,MultipleChoices,1.MolecularcloninginthefieldofDNArecombinationtechnologyiscalledas,A.theestablishmentofmonoclonalantibodiesB.theestablishmentofmultipleantibodiesC.constructionofrecombinantDNAD.asexualreproductionDNAE.SexualreproductionDNA,59,MultipleChoices,2.Thebasicconditionforatargetgenevectoris,A.thatitcanbereplicatedindependentlyB.thatithasmultipleincisionsC.thatitsmolecularweightislargeD.thatitshouldnthavegeneticmarkersE.thatitcouldntcoexistwithbacteria,60,MultipleChoices,3.Whichonecouldnotbeusedasacloningvectoris,A.plasmidDNAB.phageDNAC.bacterialgenomicDNAD.adenovirusDNAE.anti-transcriptvirusDNA,61,MultipleChoices,4.IfthefollowingsequenceiscuttedbyoneRE,5GGGGGGAATTCC3,itwouldproduce,A.5overhangendsB.3overhangendsC.5and3overhangendsD.5or3overhangendsE.bluntends,62,2.ThebasicprocessofDNAcloning,*ThepreparationoftargetDNA,*Theselectionandpreparationofvectors,*TheligationofDNAfragmentsinvitro,*ForeignDNAistransportedintohostcells,*ThescreeningandidentifyingoftargetDNA,63,(1)ThepreparationoftargetDNA,ItrepresentswholeDNAsequenceofagenome,Tofindafragmentfromgenomiclibrary,GenomiclibrarycontainsacomprehensiveDNAfragmentsfromgenomicDNAcutbyspecificRE.Duringtheconstructionofagenomiclibrary,DNAfragmentsandtheirvectorsareligated,andthenintroducedintorecipientcells.,64,TopreparefromcDNAlibraryorcDNA,ExtractingtotalmRNA,Reversetranscription,Ligation,ItrepresentsthepopulationofmRNAscodingforgenesandproteinexpression,Transformation,Proliferation,AbundantClones,65,Topreparethegenefragmentwithothermethods,a.PCRamplificationb.TosynthesizetheDNAfragmentbychemicalmethodItistypicallyusedforthoseofthesmallbiologicallyactivepeptides,66,(2)Theselectionandpreparationofvectors,PlasmidphagecosmidM13phage,Capacity10kb22kb4050kb200copies,Kallmannsyndrome(Kallmann综合征)KALIG-1genedeficientmutation,90,2.BiopharmaceuticalPreparation,Forexamples:PharmaceuticalProductsbyRecombinantDNA,91,3.GeneDiagnosisandTherapy,(1)Genediagnosis,Genetictesting(alsocalledDNA-basedtests,genediagnosis)isamongthenewestandmostsophisticatedoftechniquesusedtotestforgeneticdisorderswhichinvolvesdirectexaminationoftheDNAmoleculeitself.Othergenetictestsincludebiochemicaltestsforsuchgeneproductsasenzymesandotherproteinsandformicroscopicexaminationofstainedorfluorescentchromosomes,92,3.GeneDiagnosisandTherapy,(2)Genetherapy,GenetherapyistheuseofDNAasapharmaceuticalagenttotreatdisease.ItderivesitsnamefromtheideathatDNAcanbeusedtosupplementoraltergeneswithinanindividualscellsasatherapytotreatdisease.ThemostcommonformofgenetherapyinvolvesusingDNAthatencodesafunctional,therapeuticgeneinordertoreplaceamutatedgene.Otherformsinvolvedirectlycorrectingamutation,orusingDNAthatencodesatherapeuticproteindrug(ratherthananaturalhumangene)toprovidetreatment.,93,3.GeneDiagnosisandTherapy,(2)Genetherapy,Somaticcellgenetherapy(体细胞基因治疗)Germlinegenetherapy(性细胞基因治疗)Onlybeusedinanim