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InformationPanelOptimizationandTroubleshootinginPCR
KennethH.Roux
INTRODUCTION
Theuseofpolymerasechainreaction(PCR)togeneratelargeamountsofadesiredproductcanbeadouble-edgedsword.Failuretoamplifyunderoptimumconditionscanleadtothegenerationofmul-tipleundefinedandunwantedproducts,eventotheexclusionofthedesiredproduct.Attheotherextreme,noproductmaybeproduced.Atypicalresponseatthispointistovaryoneormoreofthemanyparametersthatareknowntocontributetoprimer-templatefidelityandprimerextension.HighonthelistofoptimizationvariablesareMg++concentrations,bufferpH,andcyclingconditions.Withregardtothelast,theannealingtemperatureismostimportant.Thesituationisfurthercomplicatedbythefactthatsomeofthevariablesarequiteinterdependent.Forexample,becausedNTPsdirectlychelateaproportionalnumberofMg++ions,anincreaseintheconcentrationofdNTPsdecreasestheconcentrationoffreeMg++availabletoinfluencepolymerasefunction.Thisarticlediscussesvariousoptimizationstrategies,includingtouchdownPCRandhot-startPCR.
RELATEDINFORMATION
ProtocolsforPCRAmplificationofHighlyGC-RichRegions(HansenandJustesen2006)andLongandAccuratePCR(Barnes2006)areavailable,asisadicussionofStrategiesforOvercomingPCRInhibition(R?dstr?metal.2008).AdetaileddiscussionofPCRprimerdesignisgiveninPCRPrimerDesign(ApteandDaniel2009).
TOUCHDOWNPCR
Touchdown(TD)PCRrepresentsafundamentallydifferentapproachtoPCRoptimization(Donetal.1991).Ratherthanmultiplereactiontubes,eachwithdifferentreagentconcentrationand/orsetofcyclingparameters,asingletube,orasmallsetoftubes,isrunundercyclingconditionsthatinher-entlyfavoramplificationofthedesiredamplicon,oftentotheexclusionofartifactualampliconsand“primer-dimers”.Multiplecyclesareprogrammedsuchthattheannealingsegmentsinsequentialcyclesarerunatincrementallylowertemperatures(see“ProgrammingtheThermalCyclerforTDPCR,”below).Ascyclingprogresses,theannealing-segmenttemperature,whichwasselectedtobeinitiallyabovethesuspectedTm,graduallydeclinesto,andfallsbelow,thislevel.Thisstrategyhelpsensurethatthefirstprimer-templatehybridizationeventsinvolveonlythosereactantswiththegreat-estcomplementarity;i.e.,thoseyieldingthetargetamplicon.EventhoughtheannealingtemperaturemayeventuallydropdowntotheTmofnonspecifichybridizations,thetargetampliconwillhavealreadybegunitsgeometricamplificationandisthusinapositiontooutcompeteanylagging(non-specific)PCRproductsduringtheremainingcycles.Becausetheaimistoavoidlow-Tmprimingdur-ingtheearliercycles,itisimperativethatTDPCRbeperformedwiththe“hotstart”modification(D’Aquilaetal.1991;Erlichetal.1991;Ruanoetal.1992)(see“Hot-StartPCR,”below).TDPCRshouldbeviewednotsomuchasamethodofdeterminingtheoptimumcyclingconditionsforaspe-cificPCR,butasapotentialone-stepmethodforapproachingoptimalamplification.Wehavefoundthatavarietyofotherwisesatisfactorysingle-amplicon-yieldingreactionsarerenderedmorerobust(i.e.,yieldmoreproduct)whensubjectedtoTDPCR(HeckerandRoux1996).
AdaptedfromPCRPrimer:ALaboratoryManual,2ndedition(eds.DieffenbachandDveksler).CSHLPress,ColdSpringHarbor,NY,USA,2003.Citeas:ColdSpringHarbProtoc;2009;doi:10.1101/pdb.ip66?2009ColdSpringHarborLaboratoryPresswww.cshprotocols.org1
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TDPCRisofparticularvaluewhenthedegreeofidentitybetweentheprimerandtemplateisunknown(Roux1994;HeckerandRoux1996).Thissituationoftenariseswhenprimersaredesignedonthebasisofaminoacidsequences,whenmembersofamultigenefamilyareamplified,orwhenevolutionaryPCRisattempted;i.e.,amplificationofDNAfromonespeciesusingprimerswithiden-titytoahomologoussegmentofanotherspecies.Insuchcases,themismatchesbetweentheprimersandtemplatemayresultinTmvaluesthataresolowthattheyapproachtheTmvaluesofthespuriousprimingsites.Degenerateprimerswithmultiplebasevariationsorinosineresiduesareoftenusedinsuchsituations(Knothetal.1988;Leeetal.1988;PatilandDekker1990;Batzeretal.1991;Petersonetal.1991),butthegreatervarietyofsequencesintheformercaseandtherelaxedstringencyinthelattercasemighttendtoincreasethechancesofnonspecificpriming.Moreover,insomecases,thelocationsofpotentialbasemismatcheswillbeunknown.AlthoughTDPCRcanbeusedwithdegen-erateprimers(Batzeretal.1991),wehaveshownthatnondegenerateprimersdisplayingasignificantdegreeoftemplate-sequencemismatchcanyieldsingle-targetampliconsofsingle-copygenesfromgenomicDNAunderstandardbufferconditions(Roux1994).Evenmismatchesclusterednearthe3′endoftheprimeraretolerated.TDPCRhastheaddedbenefitofcompensatingforsuboptimalbuffercomposition(e.g.,Mg++concentration)aswell(HeckerandRoux1996).
PROGRAMMINGTHETHERMALCYCLERFORTDPCR
ThegoalinprogrammingforTDPCRistoproduceaseriesofcycleswithprogressivelyloweranneal-ingtemperatures.Theannealingtemperaturerangeshouldspan~15°CandextendfromatleastafewdegreesabovetheestimatedTmto10°Corsobelow.Forexample,foracalculatedprimer-tem-plateTmof62°Cwithnodegeneracy,programthethermalcyclertodecreasetheannealingtemper-ature1°Ceverysecondcycle(i.e.,runtwocyclesperdegree)from65°Cto50°C,followedby15additionalcyclesat50°C.
SomethermalcyclersreadilyaccommodateTDPCRandareeasilyprogrammedtodecreasethetemperatureofasegmentautomaticallybyafixedamountpercycle(e.g.,0.5°C/cycle).Forothers,alongseriesoffilesmustbelinkedorextensivestringsofcommandsentered.Intheselattercases,itmaybemoreconvenienttocreatea“generic”TDPCRprogramcoveringabroadertemperaturerange(~20°C)thantoreprogrameverytimetherangeneedstobemodifiedbyafewdegrees.AnotheralternativeistousestepdownPCR,inwhichfewer,butmoreabruptsteps(e.g.,seven2°Cstepsorfive3°Csteps)areused(HeckerandRoux1996).
ThecontinuedpresenceofspuriousbandsfollowingTDPCRindicatesthattheinitialannealingtemperaturewastoolow,thatthereisarelativelysmallgapbetweentheTmvaluesofthetargetandunwantedamplicons,and/orthattheunwantedampliconsarebeingmoreefficientlyamplified.Raisingthenumberofcyclesper1°C-descendingsteptothreeorfourwillgivethetargetampliconanaddedcompetitiveadvantagebeforetheinitiationofthespuriousamplification.Aproportionalnumberofcyclesshouldberemovedfromtheendoftheprogramtopreventexcesscyclingandtheconcomitantdegradationoftheampliconandgenerationofhigh-molecular-weightsmears(BellandDeMarini1991).
ModificationsofTDPCRforusewithdegenerateandmismatchedprimersincludeloweringtheannealingtemperaturerange(e.g.,50°Cdecliningto35°C),whilekeepingthelast15cyclesat50°Cormore(oncepriminghasbegun,theprimersarefullycomplementarytothenewlyformedampli-cons,haveamuchhigherTm,anddonotbenefitfromexcessivelylowannealingtemperatures).
HOT-STARTPCR
EvenbriefincubationsofaPCRmixattemperaturessignificantlybelowtheTmcanresultinprimer-dimerformationandnonspecificpriming.Hot-startPCRmethods(D’Aquilaetal.1991;Erlichetal.1991;Ruanoetal.1992)candramaticallyreducetheseproblems.TheaimistowithholdatleastoneofthecriticalcomponentsfromparticipatinginthereactionuntilthetemperatureinthefirstcyclerisesabovetheTmofthereactants.Forexample,insmallerassaysincorporatinganoiloverlay,oneofthecomponentscommontoalltubes(e.g.,Taqpolymerase)canbeinitiallywithheldandaddedonlyafterthetemperaturerisesabove85°Cduringthefirstdenaturingstage.Alternatively,awaxbeadcanbemeltedoverthebulkofthereactionmixineachtubeandallowedtosolidify,andthewithheldcomponentcanbepipettedontopofthewaxcap.Thesebeadscanbemadeinthelaboratory(BassamandCaetano-Anolles1993;WainwrightandSeifert1993)orpurchased(AmpliwaxPCR
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Gems,AppliedBiosystems).Duringthetemperaturerampintothefirstdenaturationsegment,thewaxwillmeltandthefinalcomponentwillbecomeincorporatedandmixedbyconvectionineachtube,agreatconveniencewhendealingwithlargenumbersoftubes.AnothervariationonthisthemeistheuseofantibodytoTaq(TaqStartAntibodies,Clontech;JumpStartTaq,Sigma)thatbindstoandpreventsthefunctionoftheenzymeuntiltheantibodyisdenaturedbyhighheatinthefirstcycleseg-ment.OnecanalsobuyformsofTaqthatareinherentlyinactiveatlowertemperature(AmpliTaqGold,AppliedBiosystems;HotStarTaq,Qiagen)orwax-encapsulatedTaqpolymerase(TaqBeads,Promega).
OPTIMIZATIONSTRATEGY
ThestrategypresentedhereisforTDPCR,butthesameprinciplesapplytoconventionalPCR.
1.DesignoptimalprimerpairsthatarecloselymatchedinTm.Foradditionaldiscussionofprimer
design,seePCRPrimerDesign(ApteandDaniel2009).
2.CalculateorestimateapproximateTm.ProgramthethermalcyclerforTDPCR(see“Programming
theThermalCyclerforTDPCR,”above).
3.Setupseveralstandardhot-startPCRmixesincorporatingarangeofMg++concentrationsand
includingappropriatepositiveandnegativecontrols.Use104-105copiesofthetemplate.
4.Amplifyasaboveandanalyzeproductsbyagaroseoracrylamidegelelectrophoresis.Acrylamide
gelsareconsiderablymoresensitivethanagarosegels.Ampliconsthatresultedfrominefficientamplificationscanberevealedbyaprobeofthedriedgelorablot.
5.Iflittleornoproductisdetected,trythefollowingmodifications:
?Subjectreactiontubesto10additionalcyclesataconstantannealingtemperature(e.g.,55°C)andrecheck.?Reamplify10-folddilutions(1:100to1:10,000)ofinitialTDPCRatafixedannealingtempera-turefor30cycles.?Usemoretemplateandcheckforthepresenceofinhibitorsinthetemplatepreparationbyspik-ingtheoriginalPCRmixwithdilutionsofaknownpositive(demonstrablyamplifiable)template.?Add,extend,orincreasethetemperatureoftheinitialtemplatedenaturationsteppriortocycling(5minat95°Cisstandard).ThesechangeswillincreasethelikelihoodthatthetemplateDNAisfullydenaturedtoprovidethemaximalnumberofprimingsites.Anin-tubethermo-couplecanbeusedtopredeterminethattheindicatedtemperaturewillcorrespondtotheactualsampletemperature.?Varyconcentrationsofotherbuffercomponents(pH,Taqpolymerase,dNTPs,primers).?AddenhancerstothePCRmix(see“EnhancingAgents,”below).
?Reamplify10-folddilutions(1:100to1:10,000)offirstreactionusingnestedprimers.?Abandontheoriginalprimerset,designnewprimers,andbeginagain.
6.Ifmultipleproductsorahigh-molecular-weightsmearisobserved,trythefollowingmodifications:
?Raisethemaximumandminimumannealingtemperatures(i.e.,shifttherangeupward)intheTDPCRprogram.?Decreasethetotalnumberofcyclesbyeliminatingsomecyclesfromthebottomoftherangeand/orfromtheterminalconstanttemperaturecycles.?Increasethenumberofcyclesperdegreeannealingtemperaturebyonecycle,i.e.,tothreecycles/degree.Doingsomaynecessitateremovingsomelower-endand/orterminalcyclestopreventsmearingduetoexcesscycling.?Varyconcentrationsofotherbuffercomponents(pH,Taqpolymerase,dNTPs,primers).?Attemptbandpurificationfollowedbyreamplification.Targetbandscanbecutfromagarosegelsandallowedtodiffuseoutorbeliberatedbyfreeze/thawcyclesorenzymaticgeldigestion.Alternatively,asmallplugofgelcanberemovedwithamicropipettetipor,mostsimply,bystabbingthebanddirectlyinthegelwithanautoclavedtoothpickandinoculatingafreshreac-tiontube.
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?Reamplify1:104and1:105dilutionsoffirstreactionusingnestedprimers.?Abandontheoriginalprimerset,designnewprimers,andbeginagain.
MOREDETAILSABOUTSTRATEGY
VariablesthataffectPCRproductspecificityandyieldarelistedinTable1.AdditionaldiscussionofPCRoptimizationandcontaminationavoidancestrategiescanbefoundinNewtonandGraham(1994)andMcPhersonandM?ller(2000).
EnhancingAgents
Variousadditivescanbeincorporatedintothereactiontoincreasespecificityandyield(PompandMedrano1991;NewtonandGraham1994),andsomereactionsmayamplifyonlyinthepresenceofsuchadditives(PompandMedrano1991).Additivesinclude:DMSO(dimethylsulfoxide)(1%-10%)Betaine(1-2M)
Polyethyleneglycol(PEG)6000(5%-15%)Glycerol(5%-20%)Non-ionicdetergents
Formamide(1.25%-10%)
Bovineserumalbumin(10-100μg/mL)
Severaloptimizationkitsincorporatingtheseandotherenhancingagents,andavarietyofbuffers,arecurrentlymarketed.SeePCRAmplificationofHighlyGC-RichRegions(HansenandJustesen2006),LongandAccuratePCR(Barnes2006),andStrategiesforOvercomingPCRInhibition(R?dstr?metal.2008)fordiscussionsofenhancingagents.
MatrixAnalyses
Afullmatrixanalysis,inwhichseveralvaluesforeachofthevariablesaretestedincombinationwitheachoftheothervariables,canquicklybecomeoverwhelminglycumbersomeandcostly.ThesizeofthematrixcanbesignificantlypareddownbyapplyingtheTaguchimethod(Taguchi1986),inwhichseveralkeyvariablesaresimultaneouslyaltered(CobbandClarkson1994).AmoretypicalstrategyistorunasimplematrixanalysisfocusedonthoseparametersmostlikelytohavethegreatestimpactonPCRprimerhybridizationandenzymefidelity;i.e.,Mg++concentrationandannealingtemperature.
Table1.Conditionsfavoringenhancedspecificity
Usehotstart
UseTDPCR(enhancesspecificityandsensitivity)Optimizeprimerdesign↓Mg++
↓dNTP(alsofavorshigherfidelity)OptimizepH
↓Taqpolymerase
↓Cyclesegmentlengths↓Numberofcycles
↑Annealingtemperature↓Inhibitors↑Rampspeed
Addandoptimizeenhancer(s)↓Primerconcentration↓Primerdegeneracy
↑Templatedenaturationefficiency
Adjustingconditionsinthedirectionoppositethatlistedaboveusuallyfavorsincreasedsensitivity(i.e.,moreproduct)andtheconcomitantriskofnon-specificamplification.Theaimistostrikeabalancebetweenthesetwoopposingtendencies.↑and↓signifyincreaseanddecrease,respectively.
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Mg++Concentration
Mg++concentrationistheeasiesttomanipulatebecauseallconcentrationvariationscanberunsimul-taneouslyinseparatetubes.SuppliersofTaqpolymerasenowprovideMgCl2solutionseparatefromtherestofthestandardreactionbuffertosimplifyitsadjustment.Atypicaltwo-stepoptimizationseriesmightfirstincludeMg++at0.5mMincrementsfrom0.5to5.0mMand,aftertherangeisnar-rowed,asecondroundcoveredbyseveral0.2or0.3mMincrements.
AnnealingTemperature
OptimizationofannealingtemperaturebeginswithcalculationoftheTmvaluesoftheprimer-tem-platepairsbyoneofseveralmethods,thesimplestbeingTm=4(G+C)+2(A+T)forprimerslessthan21baseslong.Asingle-basemismatchlowerstheTmby~5°C.Morecomplexformulascanalsobeused(Sambrooketal.1989;Sharrocks1994),butinpractice,becausetheTmisvariouslyaffectedbytheindividualbuffercomponentsandeventheprimerandtemplateconcentrations,anycalcu-latedTmvalueshouldberegardedasanapproximation.Severalreactionsrunattemperatureincre-ments(2°C-5°C)straddlingapoint5°CbelowthecalculatedTmwillgiveafirstapproximationoftheoptimumannealingtemperatureforagivensetofreactionconditions.Itshouldbenotedthatsomeprimers,forreasonsthatarenotentirelyapparent,arerefractorytooptimization(Heetal.1994).OnepossibleexplanationmaybethatuniquecharacteristicsofthetargetamplicongiveaTmabovethetemperatureofthedenaturationcyclesegment(Sharrocks1994).Ifpermissible,itmaybemoretimeandcostefficientsimplytodesignasecondsetofprimersthathybridizetoneighboringDNA.Thermalcyclersthatgenerateauniformtemperaturegradientacrosstheheatingblockcangreatlysimplifydeterminationoftheoptimumannealingtemperature,buttheprecisethermalcharacteris-ticsyieldingtheoptimumamplificationmaybedifficulttodetermineforsomemodels.
Inhibitors
NumerousinhibitorsofPCRhavebeendescribed.Theseincludeionicdetergents(e.g.,SDSandSarkosyl;Weyantetal.1990),phenol,heparin(Beutleretal.1990),xylenecyanol,andbromophenolblue(Hoppeetal.1992).Ifinhibitorsarepresentinthetemplatepreparation,a100-folddilutionofthestartingtemplatemaysufficientlydiluteouttheinhibitor.Reextraction,ethanolprecipitation,and/orcentrifugalultrafiltrationalsomayresolvetheproblem.ProteinaseKcarryovercandigestTaqpolymerase,butisreadilydenaturedbya5-minincubationat95oC.
CycleNumber,Reamplification,andProductSmearing
Increasingthenumberofcyclesmayenhanceananemicreaction,butthismodificationcanalsoleadtothegenerationofspuriousbandsandtosmearscomposedofhigh-molecular-weightproductsrichinsingle-strandedDNA(BellandDeMarini1991).Similarsmearingcanoccurundernormalcondi-tionsifthequantityofstartingtemplatesistoogreat,asoftenoccursinattemptstoreamplifyfromapreviousPCR.Ageneralruleofthumbforreamplificationofaproductthathasbeendetectedonanagarosegelistouse1μLofa1:104to105dilutionofthePCR.
ACKNOWLEDGMENTS
IthankRaniDhanarajan,DanGarza,andKarlHeckerfortheirvaluablecomments.
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