Bio-Rad荧光定量PCR应用指南(完整版)

PCRReferencesMathewsDHetal.,Expandedsequencedependenceofthermodynamicparametersprovidesrobustpre-dictionofRNAsecondarystructure,JMolBiol288,910–940(1999)RozenSandSkaletskyHJ,Primer3onthe–386inBioinformaticsMethodsandProtocols:MethodsinMolecularBiology(KrawetzSandMisenerS,eds),HumanaPress,Totowa,NJ(2000);sourcecodeavailableat:apracticalguide,pp11–43inRNABiochemistryandBiotechnology(BarciszewskiJandClarkBFC,eds),KluwerAcademicPublishers,Boston(1999)LegalNoticesAmplifluorandUniPrimeraretrademarksofMilliporeCorporation.AmpliTaq,AmpliTaqGold,andTaqManaretrademarksofRocheMolecularSystems,Inc.BDQZymeisatrademarkofBecton,DickinsonandCompany.BeaconDesignerisatrademarkofPREMIERBiosoftInternational.BHQandBlackHoleQuencheraretrademarksofBiosearchTechnologies,Inc.CyisatrademarkofGEHealthcare.DNeasyisatrademarkofQiagenGmbHCorporation.DyNAzymeisatrademarkofFinnzymesOy.Eclipseisatrade-markofEpochBiosciences,Inc.ExcelisatrademarkofMicrosoftCorporation.HoechstisatrademarkofHoechstAktiengesellschaft.LUXisatrademarkofInvitrogenCorporation.OligoisatrademarkofMolecularBiologyInsights,Inc.PicoGreen,SYBR,andTexasRedaretrademarksofMolecularProbes,Inc.RoundupReadyisatrademarkofMonsantoCompany.ScorpionsisatrademarkofDxSLimited.VICisatrademarkofAppleraCorporation.MolecularbeaconsandtheirusearelicensedunderpatentsownedbyThePublicHealthResearchInstituteoftheCityofNewYork,Inc.Primer3includessoftwaredevelopedbytheWhiteheadInstituteforBiomedicalResearch.Bio-RadLaboratories,Inc.islicensedbyMolecularProbes,Inc.tosellreagentscontainingSYBRGreenIforuseinreal-timePCR,forresearchpurposesonly.NoticeregardingBio-Radthermalcyclersandreal-timesystems.Purchaseofthisinstrumentconveysalimitednon-transferableimmunityfromsuitforthepurchaser’sowninternalresearchanddevelopmentandforuseinappliedfieldsotherthanHumanInVitroDiagnosticsunderoneormoreofU.S.PatentsNos.5,656,493,5,333,675,5,475,610(claims1,44,158,160–163and167only),and6,703,236(claims1–7only),orcorrespondingclaimsintheirnon-U.S.counterparts,ownedbyAppleraCorporation.Norightisconveyedexpressly,byimplicationorbyestoppelunderanyotherpatentclaim,suchasclaimstoapparatus,reagents,kits,ormethodssuchas5'nucleasemethods.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingtheDirectorofLicensing,AppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404,USA.Bio-Rad'sreal-timethermalcyclersarelicensedreal-timethermalcyclersunderApplera'sUnitedStatesPatentNo.6,814,934B1foruseinresearchandforallotherfieldsexceptthefieldsofhumandiagnosticsandveterinarydiagnostics.1.PCR21.1PCR21.1.1PCR?21.1.2PCR31.1.3qPCR42.82.182.2qPCR92.2.1?92.2.292.2.2.1DNA102.2.2.2122.3SYBRGreenI192.3.1192.3.2202.3.2.1202.3.2.2222.4232.4.1232.4.2243.283.1283.2293.3293.4293.5304.PCR344.1354.1.1354.1.2354.2374.2.1374.2.2384.2.3404.2.3.12–∆∆CT(Livak)414.2.3.2∆CT424.2.3.3Pfaffl435.465.1465.2RNA475.2.1475.2.2RNA485.2.3485.3cDNA495.4qPCR505.5515.5.1515.5.2515.6526./546.1556.2TaqMan566.3DNA576.4TaqMan586.5596.6TaqMan596.7596.7.1596.7.2727.(GMO)767.1777.2DNA777.3SYBRGreenIPCR787.3.1787.3.2797.3.3797.4TaqManPCR817.4.1827.4.2827.4.3828.858.1PCR868.2868.3868.4878.5PCR888.6PCR888.7898.389PCR2PCR?2PCR3qPCR4PCR2PCRPCRPCR57PCR(GMO)1.1PCR1.1.1PCRPCRPCRDNAPCRDNAPCRDNAPCRPCRPCRDNAqPCRPCRPCRPCRPCR31.1.2PCRPCR(1.1)x-PCRy-2PCR1.128-401.1PCRCycleExponentialphaseCTvalueNon-exponentialplateauphase010203040Fluorescence0.30.20.10ThresholdlinePCR41.11-18CTCTPCRCTCTCTPCR4CT1.1.3qPCRCTPCRqPCR•R20.980r|–0.990|•90-105%•qPCRPCRDNADNAcDNAlogCTPearsonrR2qPCR1.2A2n=nCT10DNA2n=10n3.32CT3.321.2BrPCR5rR2CTrR2qPCRr0.990R20.9801.2.iCycleriQ®PCR10A.B.CTlogr95.4%ABPCR6EE=10–1/PCRPCR222210–1/–3.32E%=(E–1)x100%%=(2–1)x100%=100%1.2E=10–(1/–3.436)=1.954%=(1.954–1)x100%=95.4%1.9595.4%100%90–105%100%CTCT90%105%2.32.48qPCR999DNA1012SYBRGreenI1919202022TaqMan23232482.PCRPCR2.12.22.2.12.2.2SYBRGreenI2.3)TaqMan2.4qPCR2.1PCR••PCR••••PCRPCRPCR•EPPCR92.2qPCRPCR2.2.1PCR5••••3qPCR2.2.2qPCR21DNASYBRGreenI2TaqManEclipseAmplifluorScorpionsLUXandBDQZymePCRDNASYBRGreenITaqMan2.2.2.12.2.2.2qPCRDNA10DNA2.2.2.1DNA(SYBRGreenI)SYBRGreenIPCRDNADNAdsDNASYBRGreenIdsDNA1,0002.1dsDNAPCRUnboundSYBRGreenIBoundSYBRGreenI2.1.PCRDNAdsDNA112.2.SYBRGreenIPCRA,22PCRTmB,qPCR1,,50–2,000bp,2,3,A222PCRABdsDNA2dsDNADNATmDNA50%2.2Tm89°C23Tm78°C23DNADNAdsDNAPCRDNASYBRGreenIPCR122.2.2.2TaqManEclipseAmplifluorScorpionsLUXBDQZymeFRETPCRDNA2TaqManTaqManTaqMan2.3TaqMan5'-TaqMan5'-TaqTth5'3'/TaqTthdsDNA5'—3'FAMBHQ12.1TaqMan2.4.1TaqMan13225-40nt2.45'3'15–305-6TaqManDNA5'2.3TaqManReporterExtensionQuencherRRQQRRQQRRQQRRQQDuringannealing,theTaqManprobebindstothetargetsequenceDuringextension,theprobeispartiallydisplacedandthereporteriscleaved.Thefreereporterfluoresces14''ReporterQuencherRRQQRRQQDuringannealing,theprobebindstothetargetsequencetoseparatereporterandquencher.ThereporterfluorescesMolecularbeaconsarehairpinprobeswithreporterandquencherRRQQ2.4Donorf