FAQs

PCR

GeneralPCRGuidelines

Primers
WhatparametersdoIneedtoconsiderwhendesigningprimers?
PrimerdesignisthemostimportantfactorindeterminingthesuccessorfailureofPCRreactions.There
aretwomajorconsiderationsforprimerdesign:specificityandefficiency.

Specificityisdeterminedbythefrequencyofmisprimingevents.Primerswithpoorspecificitytendtoproduce
undesiredamplicons.
Efficiencyisdefinedastheabilityofprimerstoamplifyaproductwithatwofoldincreasepercycletothetheoretical
optimum.

Thefollowingtablesprovideguidelinesforprimerdesign.

Guidelines

Length Theoptimallengthofprimersisabout24or25bases.However,length
canbebetween21and28basesifthemeltingtemperatureneedsto
beadjusted.
WhenamplifyinglongDNAfragments(10kb),25to35merprimers
mayprovidebetterresults.

GCcontent TheGCcontent(thenumberofGsandCsintheprimerasaproportion
ofthetotalnumberofbases)shouldbe4060%.

3'end HavingfourGand/orCbasesatthe3'endmightbeusefuliftheprimer
lengthisshort(thebasesprovide"aclampingeffect"),especiallyfor
universalprimers,whicharetypicallyusedforamplifyingallcDNAor
gDNAinasample.However,addingthesebasesmayincreasenon
specificprimingeventsforgenespecificprimers.

Tm Ifpossible,designprimerswithameltingtemperature(Tm)of6870C.
Whilethisisnotabsolutelynecessary,usingstringentPCRconditions
(e.g.,"touchdownPCR"and"twostepPCR")canenhanceprimer
specificity.
 Sequence Primersshouldbespecifictoyourgeneofinterest.TheBLAST
specificity search canbeusedtofindregionsofhomology.

Note:PCRyieldoftendependsonthe3'hexamerofthePCRprimer.
Primersthatformastrongstableduplexactuallyreducethe
amplificationefficiency.

Avoid

Repeats Themaximumnumberofdinucleotiderepeatsinaprimerisfour(e.g.,
ATATATAT).

Runs Avoidlongrunsofasinglebase(morethanthree)asthiscancause
primerslippageandcontributetomispriming.

Complementary Forwardandreverseprimersshouldnotannealtoeachotherandso
3'ends shouldnothavecomplementaryGorCstretches(>4contiguous
bases).

Self Selfcomplementarity(e.g.,withintheforwardprimer)canleadto
complementary hairpinformation.AhairpinstructurecanformwithjustfourG/Cbase
3'ends pairsinthestemandthreebasesintheloop.

HowdoIcalculatethemeltingtemperatureofprimers?
Theprimermeltingtemperature(Tm)istheestimateofDNADNAhybridstability.KnowingtheTmis
criticalfordetermininganappropriateannealingtemperature(Ta).ATathatistoohighwillresultin
insufficientprimertemplatehybridization,resultinginlowPCRproductyield.ATathatistoolowmay
leadtononspecificproductamplification.
CalculationoftheTmofprimersshorterthan20basescanbeperformedusingtheWallacerule:

Tm=2C(A+T)+4C(G+C)

ForaccurateestimationoftheTmofprimerslongerthan20bases,werecommendusingfreeprimer
designsoftwaresuchasPrimer3.

WhatprimerconcentrationshouldbeusedforPCR?
Thefinalconcentrationofeachprimershouldbebetween0.1and0.5M.Astocksolutionofeachprimer
istypically1020M.

ForPCRampliconslessthan10kb,0.2Mproducessatisfactoryresults.
Foramplificationoflongtargets(~17kb)withTaKaRaLATaqDNAPolymeraseorTaKaRaExTaqDNA
 Polymerase,theprimerconcentrationcanbeincreasedupto1M.
TherecommendedprimerconcentrationforhighyieldpolymerasessuchasAdvantage2DNAPolymeraseand
TitaniumTaqDNAPolymeraseis0.4M.

Primerconcentrationsthataretoohighincreasethechanceofmispriming,whichmayresultin
nonspecificamplification.Primerconcentrationsthatarelimitingcanresultinextremelyinefficient
amplification.

HowshouldoligosbepurifiedforPCR?
StandarddesaltedprimersaresatisfactoryformostPCRapplications.

Templates
WhatistheoptimalamountoftemplateDNAthatshouldbeusedforPCR?
Theoptimalamountoftemplaterequireddependsonthecomplexityofthetemplateandthecopynumber
ofthetargetsequence.Approximately104copiesofthetargetDNAsequencearerequiredtodetectthe
amplificationproductin2530PCRcycles.

Typically,1gofhumangenomicDNAcontains3.04x105 moleculesofDNA.FormostPCRapplications,30100
ngofhumangenomicDNAissufficient.Highcopytargets,suchashousekeepinggenes,requireonly10ngof
template.Templateamountsforhighercomplexitytemplatesrangebetween10ngand500ng.
Typically,1gofE.coligenomicDNAcontains2x108 moleculesofDNAtherefore,therecommendedamountof
templateisbetween100pgand1ng.
Typically,1goflambdaDNAcontains1.9x1010 moleculesofDNAtherefore,thetemplateinputcanbeaslittle
as100pg.
TheamountofcDNAtemplatedependsonthecopynumberofthetarget.cDNAinputistypicallydescribedin
termsofequivalentRNAinput.TheamountofcDNAinaPCRreactioncanbeaslittleas10pg(RNAequivalent).

Itisimportanttonotethatnotallpolymerasescantolerateexcessiveamountsoftemplate.Forsamples
containingexcesstemplate(upto1g),werecommendPrimeSTARGXLDNAPolymerase.

Whatarecriticalfactorsforamplificationoflonggenomictargets?
Templatequality
DNAintegrityiscriticalforamplificationoflongtargets.DNAdamagesuchasDNAbreakageduring
DNAisolationorDNAdepurinationatelevatedtemperaturesandlowpHresultsinagreateramountof
partialproductsanddecreasedoverallyield.DNAdamagecanalsooccurinacidicconditionstherefore,
avoidusingwaterforresuspendingDNAtemplates.DNAismoststableatpH78orinbufferedsolutions.

PCRconditions
Denaturationtimeshouldbekepttoaminimumtodecreasedepurinationevents.
UsetouchdownPCRstartatahigherannealingtemperatureandreducebytwodegreespercycleforseveral
cycles.
Designprimerswithmeltingtemperatures(Tm)above68C.
PCRpolymerases
WeofferseveralPCRpolymerasesoptimizedforlongrangePCR.TaKaRaLATaqDNA
Polymerase,TaKaRaLATaqPolymerasewithGCBuffers,andPrimeSTARGXLDNAPolymeraseare
 recommendeddependingontheGCcontentandsizeofthetarget(s).

HowdoyoudetermineifatemplateisGCrich?
TheGCratiovariesacrossthegenome.Templateswith>65%GCcontentareconsideredGCrich.GC
richregionsofthegenomearemostlyconcentratedinregulatoryregionsincludingpromoters,enhancers,
andcisregulatoryelements.GCrichtractstendtoforminvertedrepeats,orhairpinstructures,thatmay
notmeltduringtheannealingstepofPCR.Therefore,amplificationofGCrichtemplatesishinderedby
inefficientseparationofthetwoDNAstrands.Thisresultsintruncatedampliconsduetopremature
terminationofpolymeraseextension.

WhatarethecriticalfactorsforamplificationofGCrichtemplates?
PCRconditions
Usehigherdenaturationtemperatures(e.g.,98Casopposedto94Cor95C)toallowcompletedenaturationof
thetemplate.
KeepannealingtimeforGCrichtemplatesasshortaspossible.
UseprimerswithahigherTm(>68C),becauseannealingcanbeperformedatahighertemperature.

PCRpolymerases
UseapolymeraseoptimizedforamplificationofGCrichsequences.Tofindanenzyme,visitourselectionguide.
CanDMSObeaddedtoimproveamplificationofGCrichtemplates?
WehaveheardfromcustomersthatimprovedamplificationofGCrichtemplateswasobtainedbyadding
DMSOtoreactionsusingPrimeSTARMAXDNAPolymeraseorCloneAmpHiFiDNAPolymerase.The
recommendedconcentrationofDMSOisbetween2.5%and5%.

HowcanIoptimizePCRconditionsforATrichtemplates?
SometemplatesmayhavelongATrichstretchesthatarehardtoamplifyunderstandardreaction
conditions.ThePlasmodiumfalciparumgenomeisabout80%AT,andregionsflankinggenesareoften
ATrich.

PolymerasesrecommendedforGCrichtemplates,suchasEmeraldAmpGTDNAPolymerase,
EmeraldAmpMaxDNAPolymerase,andPrimeSTARGXLDNAPolymerase,arealsosuitableforATrich
templates.

TheadvantageofhavingATrichtemplatesisthatalowerextensiontemperaturecanbeused.However,
forcertaintemplateswithATcontent>8085%,theextensiontemperaturecanbeloweredfrom72Cto
6560C.DNAreplicationatthisreducedtemperatureappearstobereliable.*

* Xinzhuan,S.,etal.(1996)ReducedExtensionTemperaturesRequiredforPCRAmplificationofExtremelyA+Trich
DNA.NuclAcidsRes.24(8):15741575.

ReactionComponents
Whatistheroleofmagnesium(Mg2+)inPCR,andwhatistheoptimalconcentration?
MagnesiumisarequiredcofactorforthermostableDNApolymerasesandisimportantforsuccessful
amplification.WithoutadequatefreeMg2+,PCRpolymerasesarenotactive.Incontrast,excessfree

Mg2+reducesenzymefidelityandmayincreasenonspecificamplification.Anumberoffactorscanaffect
 Mg2+reducesenzymefidelityandmayincreasenonspecificamplification.Anumberoffactorscanaffect
theamountoffreeMg2+inareaction,includingtemplateDNAconcentration,chelatingagentsinthe
sample(e.g.,EDTAorcitrate),dNTPconcentration,andthepresenceofproteins.

Somepolymerases(e.g.,TaKaRaExTaqDNAPolymeraseandTaKaRaLATaqDNAPolymerase)aresupplied
withamagnesiumfreereactionbufferandatubeof25mMMgCl2 .Fortheseenzymes,youcanoptimizethe
Mg2+concentrationforeachreaction.
TitaniumTaqDNAPolymeraseandAdvantage2DNAPolymerasearemagnesiumtolerantpolymerasesthatare
suppliedwithbufferscontaining3.5mMofMgCl2 .
ThefinalconcentrationofMg2+forPrimeSTARGXLDNAPolymeraseandPrimeSTARMAXDNAPolymerase
reactionsis1mMthisconcentrationincreasesfidelityfortheseenzymes.

WhatistheroleofsaltinPCRreactions?
SuccessfulPCRrequiresthattheDNAduplexseparatesduringthedenaturationstepandthatprimers
annealtothedenaturedDNA.SaltneutralizesthenegativechargesonthephosphatebackboneofDNA,
stabilizingdoublestrandedDNAbyoffsettingnegativechargesthatwouldotherwiserepeloneanother.
Potassiumchloride(KCl)isnormallyusedinPCRamplificationsatafinalconcentrationof50mM.To
improveamplificationofDNAfragments,especiallyfragmentsbetween100and1,000bp,aKCl
concentrationof70100mMisrecommended.Foramplificationoflongerproducts,alowersalt
concentrationappearstobemoreeffective,whereasamplificationofshortproductsoccursoptimallywith
highersaltconcentrations.Thiseffectislikelybecausehighsaltconcentrationpreferentiallypermits
denaturationofshortDNAmoleculesoverlongDNAmolecules.

Itisimportanttonotethatasaltconcentrationabove50mMcaninhibitTaqpolymerases.

ReactionConditions
WhenoptimizingPCRconditions,whichconditionsareparticularlyimportant?
Initialdenaturationstep

Preheatingissometimesrequiredtodenaturecomplextemplates(e.g.,genomicDNA)94Cfor1minis
sufficientfordenaturation.Excessiveheattreatmentmayleadtoenzymeinactivation.

ForTerraPCRDirectPolymeraseMix,whichisusedfordirectPCRamplificationfromtissuewithoutDNA
extractionandpurification,preheatingat98Cfor2minisrequired.
ForTaKaRaLATaqDNAPolymeraseandAdvantageGC2DNAPolymerase,aninitialdenaturationstepis
required.
PrimeSTARenzymesdonotrequirepreheatingforenzymeactivation.
Denaturingconditions
Denaturingconditionsshouldbeselectedbyconsideringthethermalcyclermodelthatwillbeused.A
generalguidelineis9495Cfor30secor98Cfor10sec.

Ifusingaheatresistantenzyme,suchasoneofthePrimeSTARpolymerases,werecommenda
denaturationstepofshortdurationandhightemperature(i.e.,510secat98C).

Denaturationatanexcessivelyhightemperatureorfortoolongmayresultinlossofenzymeactivity
and/ordamagetolongtemplates.
 Annealingconditions
Theannealingstepshouldbeadjustedforeachprimersettheannealingtemperaturedependsdirectlyon
theTmofprimers.Usingannealingtemperaturesthataretoolowmayresultinmisprimingand
nonspecificamplification,leadingtolowyieldsofthedesiredproduct.

Amplificationefficiencyandspecificitycanbeimprovedbyadjustingtheannealingtemperatureaccording
totheprimer'sTmorbyperformingtwostepPCR.

ForTaqenzymes,therecommendedannealingtimeis30sec.
EnzymesinthePrimeSTARserieshaveexcellentprimingefficiency.Therefore,itisimportanttouseashort
annealingtimeof515sec.Excessivelylongannealingtimesmayleadtomispriminginducednonspecific
amplification.
Whenamplifyingshortsequenceslessthan1kb,athreestepPCRprotocolisrecommended.ForGCrichtargets
oramplificationsoflongsequences(>10kb),atwostepPCRprotocolisrecommended.

Extensionstep
Ingeneral,anextensiontimeof1min/kbisrecommended.Whenusingthehighspeedenzymes
SpeedSTARHSDNAPolymeraseorSapphireAmpFastPCRMasterMix,useareactionrateof10
sec/kbofamplifiedproduct(i.e.,10secfora1kbproduct,20secfora2kbproduct,etc.).

PrimeSTARMaxDNAPolymeraseandPrimeSTARGXLDNAPolymerasecontainaproprietaryelongationfactorand
allowforhighspeedreactionsat520sec/kb.Ifusingtheseenzymeswithsamplescontainingexcesstemplate,an
elongationtimeof1min/kbshouldbeused.
ShouldIuseathreesteporatwostepPCRprotocol?
ThreestepPCRincludesdenaturation,annealing,andextensionsteps.Thistypeofprotocolshouldbe
usedwhentheTmoftheprimersislowerthantheextensiontemperatureorislessthan68C.

Ifthemeltingtemperatureoftheprimer(Tm)isclosetotheextensiontemperature(72C)orafew
degreeslower,considerusingatwostepPCRprotocolthatincludesadenaturationstepandacombined
annealing/extensionstep.Withthisprotocol,theannealingtemperatureshouldnotexceedtheextension
temperature.

WhichextensiontemperatureshouldIuse,68Cor72C?
A68CextensiontemperatureispreferredfortwostepPCRandwhenamplifyinglongertemplates(>4
kb).Thislowerextensiontemperaturedramaticallyimprovesyieldoflongeramplificationproductsby
reducingthedepurinationratethatinfluencesamplification.

72CshouldbeusedastheextensiontemperaturewhenperformingthreestepstandardPCRandfor
amplificationofshortfragments(<4kb).

Polymerases
HowarePCRpolymerasesshipped,andhowshouldtheybestoredandhandledafterreceipt?
TakaraClontechPCRpolymerasesareshippedondryice.Allenzymesshippedondryicewillbefrozen,
butthisfreezingprocessisgradualandonefreezethawcyclewillnotreduceenzymeactivity.Before
openinganewtubeofenzyme,spinitbrieflytocollectthecontentsatthebottomofthetube.
Polymerasesshouldbestoredat20Cinanonfrostfreefreezer(anacceptablerangeisbetween15C
and23C).Longtermstorage(longerthanaweek)at70Cor80Cisnotrecommended.Enzymes
shouldneverbeallowedtoreachroomtemperature.

HowshouldlyophilizedPCRpremixesbestored?
LyophilizedPCRpremixes,sealedintheoriginalfoilpouch,shouldbestoredatroomtemperature(20
22C).Anyunusedproductshouldalsobestoredatroomtemperature(2022C),eitherintheoriginalfoil
pouchresealedwithdesiccant,orinadesiccator.

ArethereanyspecialconsiderationswhenhandlingPCRenzymes?
Toavoidcontamination,glovesshouldbewornwhenhandlingenzymetubes,andfingersshouldbekept
awayfromthetubeopening.Itisimperativetouseanew,cleanpipettetipeverytimeyoudrawfroma
stocktubeofenzyme.

Whenpipettingenzymefromastocktube,placetheendofthetipjustfarenoughintotheliquidtoobtain
thedesiredvolume.Apipettetipshouldnotbeplungedallthewayintotheenzymesolutionasthe
outsideofthetipwillbecomecoveredwithenzyme,preventingaccuratemeasurementandwasting
enzyme.

Note:Theretentionofliquidstopolypropylenetipsvarieswithdifferenttypesofsolutions.Pipettetips
losetheirprecisionwhenliquidisdrawnmorethanonce.Lowretentionpipettetipsarerecommendedfor
usewithviscoussolutions,suchasthosecontainingglycerol.

Whatismeantbypolymerasefidelity?Whatapplicationsrequireahighfidelitypolymerase?
ThefidelityofaDNApolymerasereferstoitsabilitytoaccuratelyreplicateatemplate,oraddthecorrect
nucleotidesstartingatthe3'endoftheprimer.Therateofbasemisincorporationisknownastheerror
rate.PCRpolymeraseswithproofreadingactivitypossess3'to5'exonucleaseactivitythatcanexcise
incorrectlyincorporatednucleotidesandreplacethemwiththecorrectnucleotides.

Highfidelitypolymerasesarerecommendedforgenecloning,proteinexpression,structurefunction
studiesofproteins,cDNAlibraryconstruction,andnextgenerationsequencing.

Toselectahighfidelitypolymerase,visitthePCRSelectionGuide.

HowcanIcompareerrorratesofdifferenthighfidelitypolymerases?
Errorratesreportedbyvendorsforpolymerasescannotalwaysbedirectlycompared,asdifferent
methodsareusedtomeasurefidelity.Thesemethodsinclude:

1. Bluewhitescreening
Thisapproachisbasedonphenotypicchangesandiswidelyusedsinceitisfast,relativelysimple,andcost
effective.Theoriginalmethodforbluewhitescreening,knownastheKunkelmethod*,isbasedon
complementationofthelacZgenethatrestoresgalactosidaseenzymeactivityandallowsproductionofablue
color.Withthismethod,coloniesderivedfromlacZPCRproductscontainingsinglenucleotideerrorsor
frameshiftmutationstypicallyhaveawhitecolor,whileclonesderivedfromerrorfreeampliconsgenerateblue
colonies.
* Kunkel,T.A.andTindall,K.R.,(1987)FidelityofDNAsynthesisbytheThermusaquaticusDNA
polymerase.Biochemistry27:60086013.

2. Sequencingapproach
 ThisapproachutilizesSangersequencingofindividualcoloniesafterPCR.Thebluewhitescreeningapproach
canquicklymeasurepolymerasefidelity,howeveritisnotasaccurateasthesequencingapproach.Theblue
whitemethodwillnotdetectsilentmutations,singlenucleotidesubstitutionsthatdonotaffecttranslation.The
sequencingmethodcandetectallmutations,andthusismoreaccurate.

PrimeSTARMaxandPrimeSTARGXLDNAPolymeraseshaveveryhighfidelityhowwasfidelity
measuredfortheseenzymes?
ThefidelitiesofthePrimeSTARpolymerasesweremeasuredbySangersequencingofindividualcolonies
afterPCR.

TenarbitrarilyselectedGCrichregionsofThermusthermophilusHB8genomicDNAwereamplified.
PCRproductswereclonedintoaplasmidvector.
Multiplecloneswereselectedforeachrespectiveamplificationproduct,andthePCRinsertwassequenced.

PrimeSTARMaxDNAPolymerasehasafidelity6.5XthatofTaqpolymerase,whereasPrimeSTARGXL
DNAPolymerasehasafidelity29XthatofTaqpolymerase.

SpecialTechniques
WhatfactorsarecriticalformultiplexPCR?
AllprimerpairsusedinmultiplexPCRshouldhavesimilarprimingefficienciesfortheirtargetDNA.This
canbeachievedbyusingprimerswithnearlyidenticaloptimumannealingtemperatures.

Whendesigningprimers,payspecialattentionthefollowingparameters:

Primerhomologywiththetargetnucleicacidsequence
Length
GCcontent
Concentration
Primerhomology
(Primersshouldnothavehomologyeitherinternallyorwithoneanother,especiallyatthe3'ends.)

WhatisnestedPCR?
NestedPCRisamethodthatinvolvesreamplificationtoimprovePCRresults.NestedPCRinvolves
designinganewforwardnested(FN)orreversenested(RN)primerthatisinternaltotheoriginalprimer
andcanpairwiththeoriginalpartnerprimer.AverysmallamountoftheprimaryPCRproductisusedasa
templateforPCRwithnestedprimers.

NestedPCRfrequentlyleadstoimprovedyieldofthedesiredPCRproductby:
EliminatingextrabandsthatmayhavebeenpresentintheinitialPCR
ProducingarobustbandthatmayhavebeenweakorinvisibleintheinitialPCR

ItisimportanttonotethatonlyaverysmallamountoftheprimaryproductshouldbeusedinnestedPCR
becausethistemplatehasverylowsequencecomplexity.Tostart,theprimaryPCRproductcanbe
diluted1:100,and1lcanbeusedasthetemplatefornestedPCR.Also,youmayneedtoreducethe
numberofcyclesto2530.TheoptimalconditionsfornestedPCRshouldbedeterminedempirically.
WhatistouchdownPCR(TDPCR)andwhenwouldIneedtouseit?
Duringthedenaturationstep,allDNAmoleculeswillbecomesinglestranded.Whenthetemperature
decreasesforannealing,threetypesofduplexescanbeformed:

Homoduplexesannealingofcomplementarystrands
Heteroduplexescrosshybridizationofhomologoussequencesthatmayhavepartialhomology
Duplexesbetweenprimersandtemplate

Toachievehigherspecificity,heteroduplexformationshouldbeminimizedbyincreasingstringency(i.e.,
increasingthetemperature)duringtheinitialPCRcycles.

TouchdownPCRincreasesspecificitybyusingreactionconditionsthatgraduallyreducetheannealing
temperature.TheinitialannealingtemperatureissettoseveraldegreesabovetheestimatedTmofthe
primers.Insubsequentcycles,theannealingtemperatureisslowlydecreaseduntilitreachesthe
calculatedannealingtemperatureoftheprimers.*

ByusingahigherannealingtemperatureintheinitialPCRcycles,touchdownPCRfavorsaccumulation
ofampliconsfromsequenceswiththehighestprimertemplatecomplementarity,therebyenrichingforthe
mostspecificamplicons.Transitioningtoalowertemperatureduringsubsequentcyclesreduces
stringency,improvingprimingconditionswiththealreadyenriched,desiredtemplate.

Werecommendperforminganinitial510cycleswiththehigherannealingtemperature,andthen
graduallydecreasingthetemperatureuntiltheoptimalannealingtemperature,or"touchdown
temperature,"isreached.

Forexample,iftheTmofyourprimersis68C,therecommendedTDPCRconditionsfortheannealing
temperatureare:
5cyclesat72C,then
5cyclesat70C,then
>25cyclesat68C

* Don,R.H.,etal.(1991)'Touchdown'PCRtocircumventspuriousprimingduringgeneamplification.NuclAcids
Res.19(14):4008.

HowcanIcloneabluntendPCRproductintoaTAcloningvector?
IfaPCRproductisamplifiedwithahighfidelitypolymerasethatgeneratesbluntends,youcanperform
AtailingusingTaqpolymerase.Abriefprotocolforaddinga3'AoverhangstoaPCRproductisprovided
below.

1. PurifythePCRproduct.Beforeaddingoverhangs,itisveryimportanttoremoveallofthepolymeraseinthe
reactionbypurifyingthePCRproductusingaPCRpurificationkitorbyphenolextractionandDNAprecipitation.
Thisstepiscritical,sincetheproofreadingactivityofanyresidualDNApolymerasewoulddegradetheA
overhangs,creatingbluntendsagain.
2. PreparetheTaqDNApolymerasereactionmix:

Finalconcentration Volume(l)
 PurifiedPCRproduct 0.15to1.5pmol Varies*

dATP(10mM) 0.2mM 1

PCRBufferwithMg 1X(1.5mMMgCl2) 5
(10X)

TaqDNAPolymerase(5 1U 0.2
U/l)

ddH2O to50l

* TheAadditionreactionworksbestwhenaspecificamountofthePCRproductisused.Therecommended
amountis10100ngper100bpofthePCRproduct.Thiscorrespondsto0.151.5pmolofPCRproduct(see
tablebelow).

PCRproduct Amountof
size PCRproduct
touse

100bp 10100ng

250bp 25250ng

1,000bp 1001,000ng

3. Incubatefor20minat72C.

ProceedtoTAcloning.Foroptimalligationefficiency,it'sbesttousefreshPCRproducts,since3'A
overhangswillgraduallybelostduringstorage.

SpecialConsiderations
WhatarePCRinhibitors?
ImpuritiesthatinterferewithPCRamplificationareknownasPCRinhibitors.PCRinhibitorsarepresentin
alargevarietyofsampletypesandmayleadtodecreasedPCRsensitivityorevenfalsenegativePCR
results.PCRinhibitorsmayhavebothinorganicandorganicorigins.*

InorganicPCRinhibitorsinclude:
Calciumorothermetalionsthatcompetewithmagnesium
EDTAthatbindstomagnesium,reducingitsconcentration
SomeorganicPCRinhibitorsinclude:
Polysaccharidesandglycolipidsthatmimicthestructureofnucleicacids,interferingwithprimerbindingtothe
template
MelaninandcollagenthatformareversiblecomplexwithDNApolymerase
HumicacidsthatinteractwithtemplateDNAandpolymerase,preventingtheenzymaticreactionevenatlow
concentrations
Ureathatmayleadtothedegradationofthepolymerase
OtherorganiccompoundsthatcaninhibitPCRinclude:
Hemoglobin,lactoferrin,andIgGinblood,serum,orplasmasamples
Anticoagulantssuchasheparin
Polyphenols,pectin,andxylanefromplants
Ethanol,isopropylalcohol,phenol,ordetergentssuchasSDS

Ifinhibitorsarepresentinthetemplatepreparation,a100folddilutionofthestartingtemplatemay
sufficientlydilutetheinhibitorandallowamplification.Alternatively,ethanolprecipitationofthetemplate
maybeneededtoresolvetheproblem.

* Schrader,C.,etal.(2012)PCRinhibitorsoccurrence,propertiesandremoval.JApplMicrobiol.113:10141026.

WhatisPCRovercycling?HowdoIknowifmyproductisovercycled?
PCRovercyclingiswhencyclinggoesbeyondtheexponentialphaseofamplification.Overcyclingoccurs
whenthefollowingeventstakeplaceduringPCR:

Depletionofsubstrates(dNTPsorprimers)
Thereagents(dNTPsorenzymes)arenolongerstableatthedenaturationtemperature
ThePCRpolymeraseisinhibitedbytheproduct(pyrophosphate,duplexDNA)
Competitionforreagents(dNTPsandprimers)bynonspecificproducts
LoweringofthepHofthereaction
Incompletedenaturation/strandseparationofproductsathighproductconcentrations

TheindicatorofPCRovercyclingisanintensebackgroundsmearwithindistinguishablebandswhenthe
reactionisresolvedonanagarosegel.

ItisalwaysrecommendedtoperformapreliminarytesttodeterminetheminimalnumberofPCRcycles
neededtoyieldasufficientproduct.ThePCRproductremainsinthelinearphaseofamplificationifthe
productyieldisnoticeablyincreasedevery35cycles.

WefindthatovercycledcDNAdoesnotproducesuitabletemplateforanydownstreamapplication.

WhattypesofmutationscanbecausedbyPCR?
PCRpolymerasescanintroducedifferenttypesofmutations,includingsinglebasesubstitutions,
deletions,andinsertions.Basesubstitutionsaretypicallycausedbymisincorporationofanincorrect
dNTPduringDNAsynthesis.

Polymerasesalsogeneratemutationsatlocationswhereoneormorenucleotidesarelostorgained.The
frequencyofthistypeofmutationcanbesequencedependent,andmightbehigherinhighlyrepetitive
sequences.Themostcommonmutationisalossofasinglenucleotide,whichcouldbearesultof
templateprimermisalignmentwithinarepetitivehomopolymericsequence.

DNArearrangementscanalsooccurwhenthepolymeraseterminatessynthesisononeDNAstrandand
continuessynthesisafterprimingoccursonacomplementarystrand(i.e.,strandswitchingorjumping
PCR).ThistypeofmutationtakesplacewhenthereishighhomologybetweendifferentregionsofDNA.
ExcessiveDNAtemplateinthereactionmayalsopromotethistypeofmutation.

WhatfactorscontributetoPCRintroducedmutations?
ThefollowingfactorscancontributetoPCRintroducedmutations:

UnbalanceddNTPconcentrations
UnequalamountsofthefourdNTPscanincreasebasesubstitutionbyashighaseightfold.Usingequal
concentrationsofthefourdNTPsiscriticalforreducingtheerrorrateofpolymerase.
Highenzymeconcentration
Longincubationtimes
Lackof3'to5'exonucleaseactivity
Magnesiumconcentration
FidelityishighestwhentheconcentrationofMg2+isequimolartothetotalconcentrationofthedNTPs.Fidelity
decreaseswhentheconcentrationoffreedivalentcationsincreases.
pHofthereaction
LoweringthepHofthereactionbythreeunitscanincreasebasesubstitutionsupto60fold.LowpH(<6.0)may
leadtospontaneouspurineloss.
DNAdamage
DNAdamagecanoccurathightemperatures,possiblyincreasingtherateofmutation.Onefrequentmutationis
deaminationofcytosinetoproduceuracil.
ThepresenceofAstretchesinprimersequences
Overcycling
ErrorrateisincreasedwhentheDNAconcentrationisincreasedduringthefinalPCRcycles.Thetotalnumberof
cyclesshouldbekepttominimumtoproducethedesiredPCRproductwithouterrors.

WhatarePCRartifacts?
ThefollowingarecommonPCRartifacts:

Primerdimers
Primerdimersareformedthroughselfcomplementarityatthe3'endoftheamplificationprimers.Primerdimersare
suspectedifproductisproducedinatemplatefreereaction(negativecontrol).Toavoidprimerdimers,primers
shouldn'thavecomplementarityattheir3'ends.
ChimericPCRproducts
ChimericPCRproductscanbecausedbyincompletelyextendedtemplate.Inotherwords,singlestranded
templatethatwasnotcompletelyreplicatedduetoprematurepolymeraseterminationcanannealtopartially
homologoustemplate.ThiscreateschimericPCRproducts.Tominimizechimeras,usethefewestpossiblePCR
amplificationcycles.
PCRbias
PCRbiasoccurswhensomesequencesareamplifiedmoreefficientlythanothersduetopreferentialbindingby
 PCRprimers.Ifonesequenceisamplified10%morethananotherinonecycle,itwillbe17.4timesmore
abundantafter30cycles.ToreducePCRbias,useahighrampratebetweenthedenaturationandannealing
stepsanduselowannealingtemperatures.Longextensiontimes(>180sec)shouldbeavoided.
PCRdrift
PCRdriftisduetostochasticfluctuationintheinteractionsofPCRreagents,particularlyintheearlycycleswhena
verylowtemplateconcentrationexists.Thisartifactisobservedinmultiplexassays,wherealossofsensitivityis
causedbytheinteractionsbetweendifferentsetsofprimers.Itisimportanttocarefullydesignprimersforthese
typesofassays.
PCRgenerateshighmolecularweightproductsthatbarelymigratethroughtheagarosegel
Thereisnogoodexplanationforthisartifact.Mostresearchersassumethatthisiscausedbyovercycling,sincein
thelaterstagesofPCR,bothsingleanddoublestrandedmoleculesaccumulate.Accumulationofsuchsingle
strandedmoleculescancreateheteroduplexesbycompetingwiththeprimers.Incompletedenaturationinlater
stages,whenthereisahighconcentrationofPCRproducts,preventsDNAstrandseparation,andthusanewly
formedampliconmayremainboundtothepreviouslymadetemplate.Thisprocesscouldrepeat,trappingPCR
productsinanetworkofmolecules.

Anotherexplanationfortheoriginofhighmolecularweightsmearsisthepartialextensionoftemplates
duringinitialPCRcycles.Partialextensionscouldbegeneratedbyjumpingartifactswhenaprimeror
singlestrandedDNAannealsandextendsfromoneprimingsite,thenannealspartiallytoahomologous
segmentelsewhere(seeChimericPCRproducts,above).Partiallyextendedmoleculescanactasanew
primers,sincetheycontainafree3'OH,andcouldgeneratechimericmoleculesthatcombinetheinitial
primingsiteandthe"jump"site.

Finally,thistypeofartifactcanalsobegeneratedwhenacrudetemplateisusedforPCR.PCRproducts
amplifieddirectlyfromanimalorplanttissuescanbecometrappedincelldebris,whichpreventsthem
frommigratinginthegel.ThisproblemcanbesolvedbyProteinaseKdigestionoftheamplifiedPCR
product:

Add15lofloadingbuffercontainingProteinaseKtotheentire50lPCRreaction.
OR
Beforeloadingyoursamplesontoagel,add1loftheloadingbuffercontainingProteinaseKto4lofthePCR
reaction.

http://www.clontech.com/US/Products/PCR/Resources/PCR_FAQs