Gene-X-ppt(Gene10-基因十)--Chapter03

Chapter3MethodsinMolecularBiologyandGeneticEngineering3.1Introduction•restrictionendonuclease–AnenzymethatrecognizesspecificshortsequencesofDNAandcleavestheduplex(sometimesatthetargetsite,sometimeselsewhere,dependingontype).3.1Introduction•cloningvector–DNA(oftenderivedfromaplasmidorabacteriophagegenome)thatcanbeusedtopropagateanincorporatedDNAsequenceinahostcell.–Vectorscontainselectablemarkersandreplicationoriginstoallowidentificationandmaintenanceofthevectorinthehost.3.2Nucleases•nucleaseshydrolyzeanesterbondwithinaphosphodiesterbond.•phosphataseshydrolyzetheesterbondinaphosphomonoesterbond.FIGURE01:Thetargetofaphosphatase(a)andanuclease(b).Anendonuclease(c)andanexonuclease(d)3.2Nucleases•endonuclease–Nucleasesthatcleavephosphoesterbondswithinanucleicacidchain.–TheymaybespecificforRNAorforsingle-strandedordouble-strandedDNA.•exonuclease–Nucleasesthatcleavephosphoesterbondsoneatatimefromtheendofapolynucleotidechain.–Theymaybespecificforeitherthe5′or3′endofDNAorRNA.3.2Nucleases•RestrictionendonucleasescanbeusedtocleaveDNAintodefinedfragments.FIGURE02:Restrictionendonuclease3.2Nucleases•Amapcanbegeneratedbyusingtheoverlapsbetweenthefragmentsgeneratedbydifferentrestrictionenzymes.FIGURE03:ArestrictionmapisalinearsequenceofsitesseparatedbydefineddistancesonDNA3.3Cloning•CloningafragmentofDNArequiresaspeciallyengineeredvector.•recombinantDNA–ADNAmoleculethathasbeencreatedbyjoiningtogethertwoormoremoleculesfromdifferentsources.•ligating(orligation)–TheprocessofjoiningtogethertwoDNAfragments.3.3Cloning•subclone–Theprocessofbreakingaclonedfragmentintosmallerfragmentsforfurthercloning.•MCS(multiplecloningsite)–AsequenceofDNAcontainingaseriesoftandemrestrictionendonucleasesitesusedincloningvectorsforcreatingrecombinantmolecules.3.3CloningFIGURE04:Plasmidtransformation3.3Cloning•transformation–Theacquisitionofnewgeneticmaterialbyincorporationofaddedexogenous,nonviralDNA.•Blue/whiteselectionallowstheidentificationofbacteriathatcontainthevectorplasmidandvectorplasmidsthatcontainaninsert.FIGURE05:E.colicoloniesonagarplateswithampicillin,IPTG,andthecolorindicatorX-gal3.4CloningVectorsCanBeSpecializedforDifferentPurposesFIGURE06:Severaltypesofcloningvectorsareavailable3.4CloningVectorsCanBeSpecializedforDifferentPurposesFIGURE07:Avectorcanbeusedinbothyeastandbacteria3.4CloningVectorsCanBeSpecializedforDifferentPurposes•Shuttlevectorscanbepropagatedinmorethanonetypeofhostcell.•Expressionvectorscontainpromotersthatallowtranscriptionofanyclonedgene.3.4CloningVectorsCanBeSpecializedforDifferentPurposes•Reportergenescanbeusedtomeasurepromoteractivityortissue-specificexpression.PhotocourtesyofRobbKrumlauf,StowersInstituteforMedicalResearchFIGURE09:Amousepromotercontrolstissue-specificexpressionoflacZFIGURE10:FluorescentproteinsarepowerfulresearchtoolsCourtesyofJoachimGoedhart,MolecularCytology,SILS,UniversityofAmsterdam.3.4CloningVectorsCanBeSpecializedforDifferentPurposes•NumerousmethodsexisttointroduceDNAintodifferenttargetcells.FIGURE11:DNAcanbeintroducedintocellsinseveralways3.5NucleicAcidDetection•Hybridizationofalabelednucleicacidtocomplementarysequencescanidentifyspecificnucleicacids.•probe–Aradioactivenucleicacid,DNAorRNA,usedtoidentifyacomplementaryfragment.3.5NucleicAcidDetection•autoradiography–Amethodofcapturinganimageofradioactivematerialsonfilm.FIGURE12:AnautoradiogramofagelpreparedfromthecoloniesdescribedinFigure3.53.5NucleicAcidDetection•insituhybridization–Hybridizationofaprobetointacttissuetolocateitscomplementarystrandbyautoradiography.FIGURE13:Thefluorescentinsituhybridization(FISH)techniqueAdaptedfromanillustrationbyDarrylLeja,NationalHumanGenomeResearchInstitute().3.6DNASeparationTechniques•GelelectrophoresisseparatesDNAfragmentsbysize,usinganelectriccurrenttocausetheDNAtomigratetowardapositivecharge.AdaptedfromanillustrationbyMichaelBlaber,FloridaStateUniversity.FIGURE14:DNAsizescanbedeterminedbygelelectrophoresisFIGURE15:AgarosegelelectrophoresispatternofSV40DNAReproducedfromW.Keller,Proc.Natl.Acad.Sci.USA72(1975):2550-2554.PhotocourtesyofWalterKeller,UniversityofBasel.3.6DNASeparationTechniques•DNAcanalsobeisolatedusingdensitygradientcentrifugation.3.7DNASequencing•Chainterminationsequencingusesdideoxynucleotides(ddNTPs)toterminateDNAsynthesisatparticularnucleotides.•Primer-Asinglestrandednucleicacidmoleculewitha3′–OHusedtoinitiateDNApolymerasereplicationofapairedtemplatestrand.3.7DNASequencing•FluorescentlytaggedddNTPsandcapillarygelelectrophoresisallowautomated,high-throughputDNAsequencing.•Thenextgenerationofsequencingtechniquesaimtoincreaseautomationanddecreasetimeandcostofsequencing.InsetphotocourtesyofJanKieleczawaFIGURE17:DideoxyNTPsequencingusingfluorescenttags3.8PCRandRT-PCR•Polymerasechainreaction(PCR)permitstheexponentialamplificationofadesiredsequence,usingprimersthatannealtothesequenceofinterest.FIGURE18:Creatingacomplementarycopyofonetemplatestrandthroughdenaturationandrapidcoolinginthepresenceofexcessprimer3.8PCRandRT-PCR•RT-PCRusesreversetranscriptasetoconvertRNAtoDNAforus