clontech smart race 说明书

CLONTECHInnovativeToolstoAccelerateDiscoverySMARTTMRACEcDNAAmplificationKitUserManualPT3269-1(PR14596)Published23May2001Catalog#:K1811-1SeeListofComponentsforstorageconditionsFORRESEARCHUSEONLYCLONTECHLaboratories,Inc.(RACE)21X.CharacterizationofRACEProducts24XI.TroubleshootingGuide27XII.References34XIII.RelatedProducts35AppendixA:DetailedFlowChartof5'-RACE36AppendixB:DetailedFlowChartof3'-RACE37AppendixC:SuppressionPCRandStep-OutPCR38Protocol#PT3269-1:Additional5'-RACEsequenceobtainedwithSMARTtechnology5TableII:SettingupthepositivecontrolRACEexperiment19TableIII:Settingup5'-RACEPCRreactions21TableIV:Settingup3'-RACEPCRreactions22NoticetoPurchaser:Thisproductisintendedtobeusedforresearchpurposesonly.Itisnottobeusedfordrugordiagnosticpurposesnorisitintendedforhumanuse.CLONTECHproductsmaynotberesold,modifiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofCLONTECH.SuppressionPCRiscoveredbyU.S.Patent#5,565,340.Foreignpatentspending.ThisproductisoptimizedforuseinthePolymeraseChainReaction(PCR)coveredbypatentsownedbyHoffmann-LaRocheandF.Hoffmann-LaRoche,Ltd.(Roche).NolicenseunderthesepatentstousethePCRprocessisconveyedexpresslyorbyimplicationtothepurchaserbythepurchaseofthisproduct.AlicensetousethePCRProcessforcertainresearchanddevelopmentactivitiesaccompaniesthepurchaseofcertainreagentsfromlicensedsupplierssuchasCLONTECHLaboratories,Inc.,whenusedinconjunctionwithanauthorizedthermalcycler,orisavailablefromPerkin-ElmerCorporation.FurtherinformationonpurchasinglicensestopracticethePCRprocessmaybeobtainedbycontactingtheDirectorofLicensingatthePerkin-ElmerCorporation,850LincolnCentreDrive,FosterCity,CA94404,oratRocheMolecularSystems,Inc.,1145AtlanticAvenue,Alameda,CA94501.CLONTECHLaboratories,Inc.(RACE)usingSMARTtechnology.ThiskitintegratesourMarathonTMcDNAAmplificationKit(Chenchiketal.,1995;1996)withourSMART(SwitchingMechanismAt5'endofRNATranscript)cDNAsynthesistechnology(patentpending).ThepowerfulcombinationofMarathonandSMARTallowsyoutoisolatethecomplete5'sequenceofyourtargettranscriptmoreconsistentlythaneverbefore.Further-more,SMARTtechnologyeliminatestheneedforproblematicadaptorligationandletsyouusefirst-strandcDNAdirectlyinRACEPCR,abenefitthatmakesRACEfarlesscomplexandmuchfaster(Chenchiketal.,1998).TheSMARTRACEkitalsoincludesrecentadvancesinPCRtechnologythatbothincreasethesensitivityandreducethebackgroundoftheRACEreactions.AsaresultyoucanuseeitherpolyA+ortotalRNAasstartingmaterialforconstructingfull-lengthcDNAsofevenveryraretranscripts.CLONTECH’sSMARTtechnologyprovidesamechanismforgeneratingfull-lengthcDNAsinreversetranscriptionreactions(Zhuetal.,2001).ThisismadepossiblebythejointactionoftheSMARTIIATMoligonucleotideandPowerScriptTMReverseTranscriptase(RT).CLONTECH’sPowerScriptisavariantofMMLVRTthat,uponreachingtheendofanRNAtemplate,exhibitsterminaltransferaseactivity,adding3–5residues(predominantlydC)tothe3'endofthefirst-strandcDNA(Figure1).InSMARTtechnology,thisactivityisharnessedbytheSMARToligowhoseterminalstretchofdGresiduescanannealtothedC-richcDNAtailandserveasanextendedtemplateforRT.AfterPowerScriptRTswitchestemplatesfromthemRNAmoleculetotheSMARToligo,acompletecDNAcopyoftheoriginalRNAissynthesizedwiththeadditionalSMARTsequenceattheend.SincethedC-tailingactivityofRTismostefficientiftheenzymehasreachedtheendoftheRNAtemplate,theSMARTsequenceistypicallyaddedonlytocompletefirst-strandcDNAs.ThisprocessguaranteesthattheuseofhighqualityRNAwillresultintheformationofasetofcDNAsthathaveamaximumamountof5'sequence(TableI).Figure1.MechanismofSMARTcDNAsynthesis.First-strandsynthesisisprimedusingamodifiedoligo(dT)primer.AfterreversetranscriptasereachestheendofthemRNAtemplate,itaddsseveraldCresidues.TheSMARTIIAOligonucleotideannealstothetailofthecDNAandservesasanextendedtemplateforPowerScriptRT.5'First-strandsynthesiscoupledwith(dC)tailingbyRTPolyA+RNApolyA3'5'SMARTIIATMOligonucleotideOligo(dT)primerTemplateswitchinga