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.

NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔 迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、 产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持 业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。NEB（北京）有限公司 New England Biolabs (Beijing) LTD. NEB（北京）有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司，负责处理其在中国内地及香港地区的全部业务，同时承担部分新产品的研发工作。纽英伦生物技术（北京）有限公司成立于2001年11月，为中国生命科学研究者提供一流的产品和专业的技术服务，促进国内外生命科学学术交流，与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。