Short_et_al_1988_-_Lambda_zap

Short_et_al_1988_-_Lambda_zap - Volume 16Number 15 1988...

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Unformatted text preview: Volume 16Number 15 1988 NucleicAcidsResearch XZAP: abacteriophageXexpressionvectorwithinvivoexcisionproperties JayM.Short*, Joseph M.Fernandez, Joseph A.Sorge andWilliam D.Huse StratageneCloning Systems, 11099 NorthTorrey PinesRoad, La Jolla,CA 92037, USA ReceivedFebruary 16,1988;Revised andAcceptedJune29, 1988 ABSTRACT A lambdainsertiontypecDNAcloningvector,LambdaZAP, hasbeenconstructed. InE.colia phagemid,pBluescnptSK(-),containedwithinthevector,canbeexcisedbyf1orM13helperphage. The excisionprocesseliminatestheneedtosubcloneDNA insertsfromthelambdaphageintoaplasmidby restrctiondigestionandligation.ThisispossiblebecauseLambdaZAPincorporatesthesignalsforboth initiationandterminationofDNA synthesisfromthefIbacteriophageoriginofreplication(1). Sixof21 restrictionsitesintheexcisedpBluescriptSKpolyflinker,containedwithintheNH2-portionofthelacZ gene,areuniqueinlambdaZAP. Codingsequencesinsertedintotheserestrictionsites,inthe appropriatereadingframe,canbeexpressedfrom thelacZpromoterasfusionproteins. Thefeaturesof thisvectorsignificantlyincreasetherateatwhichclonescanbeisolatedandanalyzed. ThelambdaZAPvectorwastestedbythepreparationofachickenlivercDNA libraryandthe isolationofactinclones byscreeningwitholigonucleotideprobes. Putativeactincloneswereexcised fromthelambdavectorandidentifiedbyDNA sequencing. TheabilityoflambdaZAP toserveasa vectorfortheconstnuctionofcDNA expressionlibrarieswasdeterminedbydetectingfusionproteinsfrom clonescontainingglucocerbrosidasecDNA'susingrabbitIgGanti-glucocerbrosidaseantibodies. INTRODUCTION Bacterophagelambdahasbeenused extensivelyasavectortoclonebothcomplementaryDNA (cDNA)andgenomicDNA. TheefficiencyoflambdaphagepackagingandsubsequentinfectionofE.coli exceedstheefficiencyofplasnidtransformation100-fold. Thisincreasedefficiencygreatlyfacilitatesthe constructionofprimarylibrariescontaininggreaterthan 1x106independentclonesand, thereby,improves thelikelihoodofisolatingrareclones. Lambdaphageplaquescanbescreenedwhenplatedathigh plaquedensityandproduce little backgroundwhenscreenedwithDNA orantibodyprobes. However, subsequentcharacterizationoftheseclonedgenesisnormallyalengthyprocess. Thelargesizeof lambdaphagevectorscomplicatestherestrictionmappingandthesequenceanalysisofgenesafter cloning. Therefore,afterinitialisolationin alambdaphagevector,DNA insertsareusuallysubclonedinto plasmidvectors. Thisprocesscanbetimeconsuning. We soughttodeveloparapidandefficientinvivosystemforthetransferofDNAfromlambdato plasmidcloningvectors. A fewpotentialexcisionmechanismswereconsideredforthedevelopmentof suchalambdaphagevector,eachwithcertainadvantagesanddisadvantages. Thelambda excisionfintegrasesystemhasbeenwellstudiedandiscapableofexcisinginsertedDNA (2).Lambda vectorscontainingacolEloriginofreplicationandanantibioticresistancegeneflankedbyaftsequences whicharerecognizedbythelambdagenesxisandint,canbeexcisedtogenerateplasmidvectors containinginsertedDNA. However,theaftsignalsrequiredforintegrationandexcisionserveno useful functionintheexcisedplasrridsandtheir...
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Short_et_al_1988_-_Lambda_zap - Volume 16Number 15 1988...

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