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"RealTime" or Kinetic PCR

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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.

TheadvantageoffluorogenicprobesoverDNAbindingdyesisthatspecifichybridizationbetweenprobeandtargetisrequiredtogeneratefluorescentsignal.Thus,withfluorogenicprobes,non-specificamplificationduetomis-primingorprimer-dimerartifactdoesnotgeneratesignal.Anotheradvantageoffluorogenicprobesisthattheycanbelabeledwithdifferent,distinguishablereporterdyes.Byusingprobeslabeledwithdifferentreporters,amplificationoftwodistinctsequencescanbedetectedinasinglePCRreaction.Thedisadvantageoffluorogenicprobesisthatdifferentprobesmustbesynthesizedtodetectdifferentsequences.

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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.

Thepolymerasechainreaction(PCR)hasrevolutionizedthedetectionofDNAandRNA.Aslittleasasinglecopyofaparticularsequencecanbespecificallyamplifiedanddetected.Theoretically,thereisaquantitativerelationshipbetweenamountofstartingtargetsequenceandamountofPCRproductatanygivencycle.Inpractice,though,itisacommonexperienceforreplicatereactionstoyielddifferentamountsofPCRproduct.Thedevelopmentofreal-timequantitativePCRhaseliminatedthevariabilitytraditionallyassociatedwithquantitativePCR,thusallowingtheroutineandreliablequantificationofPCRproducts.

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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.

**Table1.PrimerandProbesSelectionGuidelinesforQuantitativeAssays.**
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.

F.SamplePreparation

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