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"RealTime" or Kinetic PCR
蚂蚁淘
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TheDNAFacilityhousesthe“real-time”orkineticPCRinstrument,theAppliedBiosystemsModel7700sequencedetectionsystem(theTaqManinstrument).Thepolymerasechainreaction(PCR)hasrevolutionizedthedetectionofDNAandRNA.Aslittleasasinglecopyofaparticularsequencecanbespecificallyamplifiedanddetected.Theoretically,thereisaquantitativerelationshipbetweenamountofstartingtargetsequenceandamountofPCRproductatanygivencycle.Inpractice,though,itisacommonexperienceforreplicatereactionstoyielddifferentamountsofPCRproduct.Thedevelopmentofreal-timequantitativePCRhaseliminatedthevariABIlitytrADItionallyassociatedwithquantitativePCR,thusallowingtheroutineandreliablequantificationofPCRproducts.Thisinstrument,therefore,nowprovidesinvestigatorswiththeabilitytoperformverysensitive,accurate,andreproducIBLemeasurementsoflevelsofgeneexpression.Inaddition,thisinstrumentcanbeusedinotherapplicationssuchasmeasuringviralload,performingallelicdiscriminationstudies,andoptimizingPCRconditions.
A.Real-TimePCRChemistry
Real-timesystemsforPCRwereimprovedbyprobe-based,ratherthanintercalator-based,PCRproductdetection.Theprincipaldrawbacktointercalator-baseddetectionofPCRproductaccumulationisthatbothspecificandnonspecificproductsgeneratesignal.Analternativemethod,the5"nucleaseassay,providesareal-timemethodfordetectingonlyspecificamplificationproducts.Duringamplification,annealingoftheprobetoitstargetsequencegeneratesasubstratethatiscleavedbythe5"nucleaseactivityofTaqDNApolymerasewhentheenzymeextendsfromanupstreamprimerintotheregionoftheprobe.Thisdependenceonpolymerizationensuresthatcleavageoftheprobeoccursonlyifthetargetsequenceisbeingamplified.
Thedevelopmentoffluorogenicprobesmadeitpossibletoeliminatepost-PCRprocessingfortheanalysisofprobedegradation.Theprobeisanoligonucleotidewithbothareporterfluorescentdyeandaquencherdyeattached.Whiletheprobeisintact,theproximityofthequenchergreatlyreducesthefluorescenceemittedbythereporterdyebyFörsterresonanceenergytransfer(FRET)throughspace.Probedesignandsynthesishasbeensimplifiedbythefindingthatadequatequenchingisobservedforprobeswiththereporteratthe5"endandthequencheratthe3"end.Figure1diagramswhathappenstoafluorogenicprobeduringtheextensionphaseofPCR.Ifthetargetsequenceispresent,theprobeannealsdownstreamfromoneoftheprimersitesandiscleavedbythe5"nucleaseactivityofTaqDNApolymeraseasthisprimerisextended.Thiscleavageoftheprobeseparatesthereporterdyefromquencherdye,increasingthereporterdyesignal.Cleavageremovestheprobefromthetargetstrand,allowingprimerextensiontocontinuetotheendofthetemplatestrand.Thus,inclusionoftheprobedoesnotinhibittheoverallPCRprocess.Additionalreporterdyemoleculesarecleavedfromtheirrespectiveprobeswitheachcycle,effectinganincreaseinfluorescenceintensityproportionaltotheamountofampliconproduced.
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B.Instrumentation
TheABIPRISM7700SequenceDetectionSystemisaflexiblesystemdesignedtotakefulladvantageofthebenefitsoffluorogenicprobedetection.The7700systemhasabuilt-inthermalcyclerandalaserdirectedviafiberopticcablestoeachofthe96samplewells.ThefluorescenceemissiontravelsbackthroughthecablestoaCCDcameradetector.Becauseeachwellisirradiatedsequentially,thedimensionsoftheCCDarraycanbeusedforspectralresolutionofthefluorescentlight.Becausethe7700instrumentdetectsanentirefluorescencespectrum,thesystemiscapableofdistinguishingandquantitatingmultiplefluorophoresineachsamplewell.Thesoftwareanalyzesthedatabyfirstcalculatingthecontributionofeachcomponentdyetotheexperimentalspectrum.Eachreportersignalisthendividedbythefluorescenceofaninternalreferencedye(ROX)inordertonormalizefornon-PCRrelatedfluorescencefluctuationsoccurringwell-to-wellorovertime.Theuseofthisinternalreferencedye,enabledbytheabilitytodistinguishfluorophores,increasestheprecisionofthedataobtainedwiththe7700system.ThefluorescenceemissionsofSYBRGreenIdyeandROXdyearewellresolved,sothebenefitofusinganinternalreferencedyeisobtainedforSYBRGreenIdyedetectionofPCRonthe7700system.TheotheradvantageofdistinguishingfluorophoresisthatprobeslabeledwithdifferentreporterdyescanbeusedsothatmorethanonePCRtargetcanbedetectedinasingletube.
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C.Real-TimePCRQuantitation
Theabilitytomonitorthereal-timeprogressofthePCRcompletelyrevolutionizesthewayoneapproachesPCR-basedquantificationofDNAandRNA.ReactionsarecharacterizedbythepointintimeduringcyclingwhenamplificationofaPCRproductisfirstdetectedratherthantheamountofPCRproductaccumulatedafterafixednumberofcycles.Thehigherthestartingcopynumberofthenucleicacidtarget,thesoonerasignificantincreaseinfluorescenceisobserved.Figure2showsarepresentativeamplificationplotanddefinesthetermsusedinthequantificationanalysis.Anamplificationplotistheplotoffluorescencesignalversuscyclenumber.IntheinitialcyclesofPCR,thereislittlechangeinfluorescencesignal.Thisdefinesthebaselinefortheamplificationplot.AnincreaseinfluorescenceabovethebaselineindicatesthedetectionofaccumulatedPCRproduct.Afixedfluorescencethresholdcanbesetabovethebaseline.TheparameterCT(thresholdcycle)isdefinedasthefractionalcyclenumberatwhichthefluorescencepassesthefixedthreshold.AplotofthelogofinitialtargetcopynumberforasetofstandardsversusCTisastraightline.QuantificationoftheamountoftargetinunknownsamplesisaccomplishedbymeasuringCTandusingthestandardcurvetodeterminestartingcopynumber.TheentireprocessofcalculatingCTs,preparingastandardcurve,anddeterminingstartingcopynumberforunknownsisperformedbythesoftwareofthe7700system.
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D.PrimerandProbeDesign
PrimerExpresssoftwareusesasetofdefaultparameterstoautomaticallyselectprimerandprobesets.AsummaryoftheprimerandprobedesignguidelinesisshowninTable1.EventhoughnoprobeisrequiredforSYBRGreenIdyedetection,itisstillagoodideatousePrimerExpresssoftwaretoselectaprimerandprobesetwhendesigningaSYBRGreenIassay.Althoughnoprobewillbeused,theprimerswillmeetalltherequiredcriteriaandif,inthefuture,thereistheneedtoconverttheassaytoTaqManassaychemistrytoobtainhigherspecificity,theprobecanimmediatelybefoundintheoriginalPrimerExpresssoftwaredocument.
ThePrimerExpresssoftwareisloadedonaMacintoshcomputerlocatedintheDNAFacility.Inordertousetheprogram,investigatorswillneedtobringtheirtargetsequence,inatextfileformat,ona1.4Mbfloppydisk.DNAFacilitypersonnelareavailabletoprovideadviceontheuseofthesoftware.
AnimportantdefaultparameterinPrimerExpresssoftwareistheselectionofampliconsinthe50–150basepairrange.Smallampliconsarefavoredbecausetheypromotehigh-efficiencyassaysthatworkthefirsttime.Inaddition,high-efficiencyassaysenablerelativequantificationtobeperformedusingthecomparativeCTmethod(DDCT).Thismethodincreasessamplethroughputbyeliminatingtheneedforstandardcurveswhenlookingatexpressionlevelsofatargetrelativetoareferencecontrol.
Wheneverpossible,primersandprobesshouldbeselectedinaregionwithaG/Ccontentof20–80%.RegionswithaG/Ccontentinexcessofthismaynotdenaturewellduringthermalcycling,leadingtoalessefficientreaction.Inaddition,G/C-richsequencesaresusceptibletonon-specificinteractionsthatmayreducereactionefficiencyandproducenon-specificsignalinSYBRGreenIassays.Forthissamereason,primerandprobesequencescontainingrunsoffourormoreGbasesshouldbeavoided.A/T-richsequencesrequirelongerprimerandprobesequencesinordertoobtaintherecommendedTms.Thisisrarelyaproblemforquantitativeassays;however,probesapproaching40basepairscanexhibitlessefficientquenchingandproducelowersynthesisyields.
TaqManProbeGuidelines | SequenceDetectionPrimerGuidelines(SYBRGreenorTaqManAssays) |
| Selecttheprobefirstanddesigntheprimersascloseaspossibletotheprobewithoutoverlappingit(ampliconsof50–150basepairsarestronglyrecommended) | Selecttheprobefirstanddesigntheprimersascloseaspossibletotheprobewithoutoverlappingit(ampliconsof50–150basepairsarestronglyrecommended) |
| KeeptheG/Ccontentinthe20–80%range | KeeptheG/Ccontentinthe20–80%range |
| Avoidrunsofanidenticalnucleotide.Thisisespeciallytrueforguanine,whererunsoffourormoreGsshouldbeavoided | Avoidrunsofanidenticalnucleotide.Thisisespeciallytrueforguanine,whererunsoffourormoreGsshouldbeavoided |
| WhenusingPrimerExpresssoftwaretheTmshouldbe68–70°C | WhenusingPrimerExpresssoftwaretheTmshouldbe58–60°C |
| NoGonthe5´end | Thefivenucleotidesatthe3´endshouldhavenomorethantwoGand/orCbases |
| SelectthestrandthatgivestheprobemoreCthanGbases |
SelectingprimersandprobeswiththerecommendedTmsisoneofthefactorsthatallowstheuseofuniversalthermalcyclingparameters.HavingtheprobeTm8–10°Chigherthanthatoftheprimersensuresthattheprobeisfullyhybridizedduringprimerextension.
PrimerExpresssoftwaredoesnotselectprobeswithaGonthe5´end.ThequenchingeffectofaGbaseinthispositionwillbepresentevenafterprobecleavage.Thiscanresultinreducednormalizedfluorescencevalues(DRn),whichcanimpacttheperformanceofanassay.HavingGbasesinpositionsclosetothe5´end,butnotonit,hasnotbeenshowntocompromiseassayperformance.AnotherempiricalobservationisthatprobeswithmoreCthanGbaseswilloftenproduceahigherDRn.SincePrimerExpresssoftwaredoesnotautomaticallyscreenforthisfeature,itmustbecheckedmanually.IfaprobeisfoundtocontainmoreGthanCbases,thecomplementoftheprobeselectedbyPrimerExpresssoftwareshouldbeused,ensuringthataGisnotpresentonthe5´end.
Thelastfivebasesonthe3´endoftheprimersshouldcontainnomorethantwoCand/orGbases,whichisanotherfactorthatreducesthepossibilityofnon-specificproductformation.Undercertaincircumstances,however,suchasaG/C-richtemplatesequence,thisrecommendationmayhavetoberelaxedtokeeptheampliconunder150basepairsinlength.Itshould,however,befollowedasoftenaspossible,andevenwhenitisnotpossible,primer3´endsextremelyrichinGand/orCbasesshouldbeavoided.
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E.ThermalCyclingParameters
AllquantitativeassaysdesignedusingAppliedBiosystems’guidelinescanberunusingthesameuniversalthermalcyclingparameters.Thiseliminatesanyoptimizationofthethermalcyclingparametersandmeansthatmultipleassayscanberunonthesameplatewithoutsacrificingperformance.ThisbenefitiscriticalwhencombiningtwoassaysintoamultiplexTaqManassaysystem,inwhichtheoptiontoruntheassaysunderdifferentthermalcyclingparametersisnotavailable.Table2showstheuniversalthermalcyclingparametersforquantitativeTaqManorSYBRGreenIassayswhenusingDNAorCDNAasthesubstrate.
Theuserisresponsibleforsettinguptheirownreactions.ThesamplesshouldbesuppliedtotheDNAFacilityintubesorplatesthatarereadytobeplacedintotheinstrument.Inotherwords,theuserisresponsibleforacquiringthereactionreagents,primers,probe,andreactionvessel.AppliedBiosystemsTaqManreagentscanbeobtainedfromtheEnzymeCoreattheHybridomaFacility(2ndFloorEMRB).TheDNAFacilitywillberesponsibleforsettinguptheinstrument,datacollection,andpreliminarydataanalysis.
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G.DataandAnalysis
Resultswillbeavailableintwoforms.ThefirstisahardcopyoftheExperimentalReportasprovidedbytheSequenceDetectionSystemAnalysisSoftwarev.1.7.Theoutputispresentedinatabularforminwhicheachrowcorrespondstoinformationgeneratedforeachwellposition.Foreachwell,informationregardingthesamplename,CTvalue,andquantityorcopynumberisprovided.Ifastandardcurvewasrun,itsslope,fit(R),andy-interceptisprovidedintheheaderinformation.Alternatively,thesedatacanbeprovidedinanelectronicformatasaMicrosoftExcelworkbookfile.Additionaldataanalysis,suchasDDCTcalculations,istheresponsibilityoftheuser.However,corepersonnelareavailabletoassistand/oradviseusersinperformingtheseadditionalcalculations.
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H.SupplementalInformation
- Real-timePCRreviewarticle(2000)(RealtimeRevarticle.pdf)
- Real-timePCRreviewarticle(2002)(Bustin_realtimereview_2002.pdf)
- Real-timePCRoverview(realtimeoverview.pdf)
- BasicsofusingReal-timePCR(realtimePCRbasics.pdf)
- Genequantificationcalculations(genequant.pdf)
- Relativequantitationofgeneexpression(Compar_Anal_Bulletin2.pdf)
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