Description:

 

Rapid,sensitiveandpreciseprobe-basedqPCRdetectionandquantitationoftargetDNAandCDNAsequences.

Probe-basedquantitativePCR(qPCR)usesreal-timefluorescencereleasedupon5´→3´exonucleasecleavageofaquenched,target-specificprobetomeasureDNAamplificationateachcycleofaPCR.Atapointwherethefluorescencesignalissignificantlydetectableoverthebackgroundfluorescence,aquantificationcycleorC_qvaluecanbedetermined.C_qvaluescanbeusedtoevaluaterelativetargetabundancebetweentwoormoresamplesortocalculateabsolutetargetquantitiesinreferencetoanappropriatestandardcurve,derivedfromaseriesofknowndilutions.

TheNEBLunaUniversalProbeqPCRMasterMixisa2Xreactionmixoptimizedforreal-timeqPCRdetectionandquantitationoftargetDNAsequencesusinghydrolysisprobes.ItcontainsHotStartTaqDNAPolymeraseandhasbeenformulatedwithauniquepassivereferencedyethatiscompatIBLeacrossavarietyofinstrumentplatforms(includingthosethatrequireahighorlowROXreferencesignal).ItalsofeaturesdUTPforcarryoverpreventionandanon-fluorescent,visibledyetomonitorreactionsetup.ThisdyedoesnotspectrallyoverlapfluorophorescommonlyusedforqPCRandwillnotinterferewithreal-timedetection.

Themastermixformulationissuppliedat2XconcentrationandcontainsallPCRcomponentsrequiredforamplificationandquantitationofDNAexceptprimers/probesandDNAtemplate.GenomicDNAorcDNAofinterestcanbequantitatedwithLunaqPCRandexistingaswellascommercialqPCRassayprimer/probesequencescanbeused.

Figure1:NEB’sLunaUniversalProbeqPCRMasterMixoffersexceptionalsensitivity,reproducibilityandqPCRperformance

Figure2:NEB’sLunaUniversalProbeqPCRMasterMixoffersrobustperformanceinmultiplexapplications

Figure3:Extensiveperformanceevaluationofcommerciallyavailableprobe-basedqPCRreagentsdemonstratestherobustnessandspecificityofLuna

Notes:

AssayDesignTheuseofqPCRprimerdesignsoftware(e.g.,Primer3)maximizesthelikelihoodofamplificationsuccesswhileminimizingnonspecificamplificationandprimerdimers.TargetswithbalancedGC/ATcontent(40–60%)tendtoamplifyefficiently.Wherepossible,entersufficientsequencearoundtheareaofinteresttopermitrobustprimerdesignandusesearchcriteriathatpermitcross-referenceagainstrelevantsequencedatabases(toavoidpotentialoff-targetamplification).ForcDNAtargets,itisadvisabletodesignprimersacrossknownsplicingsitesinordertopreventamplificationfromgenomicDNA.Conversely,primersdesignedtotargetintronicregionscanensureamplificationexclusivelyfromgenomicDNA.PrimerandProbeConcentrationFormosttargets,afinalconcentrationof400nMforeachprimerwillprovideoptimumperformance.Ifneeded,primerconcentrationscanbeoptimizedbetween200–900nM.Probeshouldbeincludedat200nMforbestresults.Probeconcentrationcanbeoptimizedintherangeof100–500nMifoptimizationofperformanceortargetfluorescencelevelisdesired.MultiplexingTodetectorquantitatemultipletargetsinthesameLunareaction,selectdifferentfluorophorescorrespondingtoseparatedetectionchannelsofthereal-timeinstrument.Include400nMofforwardandreverseprimerand200nMprobeforeachtargettobedetectedinthereaction,andadjustconcentrationsifnecessarybasedonperformance(primer200–900nM,probe100–500nM).WhenloADIngqPCRprotocolontothereal-timeinstrument,besuretoselecttheappropriateopticalchannels,assomeinstrumentshaveasinglechannelrecordingmodethatwouldpreventmultiplexdatacollectionandanalysis.ForROX-dependentinstruments,avoidROX-labeledprobes.Thefunctionalityoftheprimerandprobesetsshouldbetestedindividuallybeforeattemptingamultiplexreaction.Whendeterminingwhichfluorophorestoincludeinamultiplexreaction,besuretochoosecompatiblereporterdyesandquenchersthathavewellseparatedfluorescencespectraorexhibitminimaloverlap.AmpliconLengthToensuresuccessfulandconsistentqPCRresults,itisimportanttomaximizePCRefficiency.AnimportantaspectofthisisthedesignofshortPCRamplicons(typically70–200bp).Someoptimizationmayberequired(includingtheuseoflongerextensiontimes),fortargetsthatexceedthatrange.TemplatePreparationandConcentrationLunaqPCRiscompatiblewithDNAsamplespreparedthroughtypicalnucleicacidpurificationmethods.PreparedDNAshouldbestoredinanEDTA-containingbuffer(e.g.,1XTE)forlong-termstability,anddilutionsshouldbefreshlypreparedforaqPCRexperimentbydilutionintoeitherTEorwater.Generally,ausefulconcentrationofstandardandunknownmaterialwillbeintherangeof106copiesto1copy.ForgDNAsamplesfromlargegenomes(e.g.,human,mouse)arangeof50ng–1pgofgDNAistypical.Forsmallgenomes,adjustasnecessaryusing106 –1copyinputasanapproximaterange.Notethatforsinglecopydilutions,somesampleswillcontainmultiplecopiesandsomewillhavenone,asdefinedbythePoissondistribution.ForcDNA,usetheproductofareactioncontaining1μg–0.1pgstartingRNA.cDNAdoesnotneedtobepurifiedbeforeadditiontotheLunareactionbutshouldbedilutedatleast1:10intotheqPCR.ROXReferenceDyeSomereal-timeinstrumentsrecommendtheuseofapassivereferencedye(typicallyROX)toovercomewell-to-wellvariationsthatcouldbecausedbybubbles,smalldifferencesinvolume,andautofluorescencefromdustorparticulatesinthereaction.TheLunaUniversalProbeqPCRMasterMixisformulatedwithauniversalreferencedyethatiscompatiblewithavarietyofqPCRinstrumenttypes,includingthosethatusenopassivereferencenormalizationandthosethatusealoworhighconcentrationofpassivereferencedye(ROX).Therefore,noadditionalcomponentsarerequiredtoensurecompatibilitywiththeseinstruments.CarryoverContaminationPreventionqPCRisanextremelysensitivemethod,andcontaminationinnewqPCRassayswithproductsfrompreviousamplificationreactionscancauseavarietyofissuessuchasfalsepositiveresultsandadecreaseinsensitivity.Thebestwaytopreventthis“carryover”contaminationistopracticegoodlaboratoryproceduresandavoidopeningthereactionvesselpostamplification.However,toaccommodatesituationswhereadditionalanti-contaminationmeasuresaredesired,theLunaUniversalProbeqPCRMasterMixcontainsamixtureofdUTP/dTTPthatresultsintheincorporationofdUintotheDNAproductduringamplification.PretreatmentofqPCRexperimentswithuracilDNAglycosylase(UDG)willeliminatepreviously-amplifieduracil-containingproductsbyexcisingtheuracilbasetoproduceanon-amplifiableDNAproduct.TheuseofaThermolabileUDGisimportant,ascompleteinactivationoftheUDGisrequiredtopreventdestructionofnewlysynthesizedqPCRproducts.Toenablecarryoverprevention,0.025units/μlAntarcticThermolabileUDG(NEB#M0372)shouldbeaddedtothereactionmix.Tomaximizeeliminationofcontaminatingproducts,setuptheqPCRexperimentsatroomtemperatureorincludea10minuteincubationstepat25°Cbeforetheinitialdenaturationstep.ReactionSetupandCyclingConditionsDuetothehotstartnatureofthepolymerase,itisnotnecessarytopreheatthethermocyclerpriortouseorsetupreactionsonice.For96-wellplates,werecommendafinalreactionvolumeof20μl.For384-wellplates,afinalreactionvolumeof10μlisrecommended.Whenprogramminginstrumentcyclingconditions,ensureaplatereadisincludedattheendoftheextensionstep,andadenaturation(melt)curveaftercyclingiscompletetoanalyzeproductspecificity.Amplificationfor40cyclesissufficientformostapplications,butforverylowinputsamples45cyclesmaybeused.

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