Complete PCR Guide

Inthepolymerasechainreaction(PCR),aThermostableDNApolymeraseamplifiesDNAthatisflankedbyknownsequences.Theknownsequencescorrespondtothoseonsyntheticoligonucleotideprimerswhichareusedtoinitiatethereaction.PCRcanbeusedinmanycomplexwaystoachievedifferentresults.GeneralIssuestoConsider:BecausePCRisverysensitive,productcontaminationbyundesiredsequencesisproblematic.Everyeffortmustmadetokeepthetemplatefreeofcontaminating,especiallypreviouslyamplified,sequences.LeavetheDNAintherefrigeratoruntilalltheotherreagentsaremixedandaliquotedinthereactiontubes.WeargloveswhenhandlingDNAtemplateandPCRreactionmixtures.Itisusuallyeasiertomakeamastermixofeverything(includingtheenzyme-itsthermalstable)exceptthetemplate.Aliquotthemixtureintotubes,andaddthetemplatejustpriortoputtingtheminathermalcycler.Otherprecautionsincludetheuseofcottonpluggedpipettipstoreduceaerosolcontaminantsofamplifiedproducts,andseparatinginitialtemplatesfromamplifiedDNAinseparaterooms.Negativetemplatecontrolsshouldbeincludedinallamplifications.OneofthemostcriticalfactorsforsuccessfulamplificationofDNAisthemagnesiumionconcentration.ToomuchMgCl₂willcausehighlevelsofnon-specificamplification,whiletoolittlewillinhibitthereaction.Thisprotocoluses1.5mMMg++asthefinalconcentrationforTaqDNApolymerase.Usethisasastartingpointwhenusingpreviouslyuntestedprimersandtemplates.Optimalconcentrationsmayvaryfrom0.5to6mMandaredeterminedbytitrationwithMgCl2.Becauseofthisvariation,10XstocksolutionsfrequentlyhavenomagnesiuminthemandtheMgCl2isaddedseparately.Generally,thedNTPandMgCl2concentrationsaresimultaneouslyadjusted.Annealingtemperaturesarecriticalandmayalsorequireexperimentaloptimization.Lowtemperatureannealingincreasesnon-specificamplification;hightemperaturesinhibitannealingbutmayincreasespecificity.Typicalreactionsareperformedinarangeof55°to65°C.ThetemperatureoptimumforTaqDNApolymeraseis72°C.ChoiceofPrimers:Anumberofconsiderationsgointodesigningprimers.Primersshouldnotbeself-complementaryorcomplementarytoeachother,especiallyattheir3endstoavoidprimer-dimersfromforming.KeeptheG+Ccontentbetween40and60%.AvoidlongstretchesofG+Csincetheymayformsecondarystructures.Additionofrestrictionenzymesitestothe5endoftheprimersserveasusefulvehiclesforsubsequentsubcloning.Primerconcentrationsshouldbeinexcessofthetemplatethroughoutthecycling.Typicallytheprimersareusedovera0.1-1.0µMrange.Lowerconcentrationsmayreduceartifactsandformationofprimer-dimers.ChoiceofTemplates:Wehavesuccessfullyusedavarietyofdifferenttemplatesinamplificationreactions.MosthumanDNApreparationsarefromfreshperipheralbloodleukocytesorcelllines.Thecellsarelysed,treatedwithproteanase-K,RNAase-treated,andphenolextracted.ThefinalA260:280ratioisabout1.8.Foramplificationofportionsofplasmids,wehaveusedeverythingfromCsCl-bandedDNA,quickprepDNA,andheatdenatured,transformedbacteria.Inthelattercase,asinglebacterialcolony(orassmallaportionofafrozenstockaspossIBLe)wasaddedto0.5mlofTE,vortexed,andthencentrifugedtopelletthebacteria.ThepelletwasresUSPendedorwashedoncein100µlofTE,centrifugedagain,andresuspendedin10µlofTE.TheentiresamplewasaddedastheDNAtemplatetoatotal50µlreactionvolume.ChoiceofDNAPolymerases:TaqDNAPolymerase-Taqhas5to3exonucleaseactivity,butneither3to5exonucleasenoranyendonucleaseactivity.Itshalf-lifeat95°Cis35-40minutes,andis10minutesat97.5°C.Molecularweight=94,000bySDS-PAGE.GeneAmp10XPCRbufferII"fromPerkin-ElmerCetusis500mMKCland100mMTris-HCl(pH8.3).BufferIhad15mMMgCl2addedtoit.Wedilutethe10XbufferinH2O;Perkin-Elmerrecommends0.15%NP-40,0.15%tween-20,0.1mMEDTAand25mMTris-HCl,pH8.3forAmpliTaqbuffer.Stoffelfragment-TheN-terminal289aminoacidsofTaqwereremovedtoreducethe5to3exonucleaseactivity.Itshalf-lifeathightemperaturesisabouttwicethatofTaqDNApolymerase.MgCl2concentrationsaretypically2-10mMgivingitabroaderrangeofmagnesiumthanTaq.TheenzymeisrecommendedforallelespecificPCR,andforamplificationofregionswithhighGCcontent.rTthDNAPolymerase-Thispolymeraseisusedinreversetranscription-PCRwhereCDNAisfirstsynthesizedat55°-70°Cinthepresenceofmanganese.ThebuffercontainsDMSOandglycerol.Oneversionofthisenzyme(rTthDNApolymeraseXL)hasproofreADIngactivityviaits3to5exonucleaseactivityandisusedtogeneratelongerPCRproducts.Promegastatesthatpolymeraseswithproofreadingactivitiesrequirehigherprimerconcentrations(0.1-0.5µM).Thehalf-lifeofrTthDNApolymeraseisabouthalfthatofTaqDNApolymerase.UlTmaDNAPolymerase-has3to5exonuclease,butnot5to3exonucleaseactivity.Itshalf-lifeat97.5°Cis40-50minutes.10XBufferfromPerkin-Elmerforthisenzymeis100mMTris-HCl,pH8.8,100mMKCl,and0.02%tween20.Itisusedat2-6U/100µlreactionvolume.Itsfidelitymaybehigherthanotherenzymes.SettinguptheReaction:ForaSingle100µlPCRReaction:10xPCRbuffer10.0µldNTPmixture(1.25mMeachdNTP)16.0µl(finalconcentration=200µMeach)5primer(20µM)5.0µl(finalconcentration=1.0µM)3primer(20µM) 5.0µl(finalconcentration=1.0µM)TaqDNApolymerase(5U/µlstock)0.5µl(2.5units)DNAtemplate varies(0.1to1.0µgforgenomicDNA;1ngorlessforclonedoramplifiedDNA)SterileWaterto100µlLayer50-75µlofmineraloilontopofreactionmixture.Samplevolumesvarybetween25and100µl.Smallervolumesarepossible,butlargervolumesmayreduceyields,perhapsbecauseoftheadditionaltimeneededtobringthesampletopropertemperature.Variationintheconcentrationofsomeofthecomponentsisacceptable.Forexample,wehavesuccessfullyamplified300bpregionswith25µMdNTPs.Primerconcentrationsmayalsobereducedto0.1µM.Asdescribedabove,a"mastermix"maybepreparedforamplificationofDNAinmultipletubes.Thefollowingtablegivesvolumes(inµl)forpreparationofamastermixturefor1through10reactionsof25µlpertube.Aslightexcessispreparedtoprovidesufficientvolumesforpipeting.CyclingParameters:Toreducenonspecificamplification,increasetheannealingtemperaturein3to5°Cincrements.Wehaveusedtheextensiontemperaturewithoutaseparateannealingtemperatureforparticularamplificationswithgoodresults."Hotstarts"refertotheadditionofthepolymeraseaftertheDNAhasdenatured.Thisshouldresultinminimizingnonspecificprimingatlowtemperatures.ThisisaccomplishedbyheatingthesampleabovethedenaturingtemperatureandthenaddingtheTaqDNApolymerasethroughthemineraloillayerintotheaqueoussolutioncontainingtheDNA.Waxplugsarealsocommerciallyavailable.Theyaremeltedabovethesample(lackingpolymerase)thenallowedtohardenatroomtemperature.Additionalreagentscanbeaddedabovethesolidwax(i.e.Taqpolymerase),andthentheentiresampleisaddedtothethermalcycler.AstheDNAisdenaturedandthewaxmelts,theenzymetransferstothesampletobeginamplification.Atypicalcyclingtemperatureprofileis:94°Cfor1minute60°Cfor1minute72°Cfor1minuteWeusea5minutestimeperiodat94°Ctodenaturetemplatepriortotheinitialcycle.Anoldruleofthumbfora72°Cextensiontemperatureis1minuteforeach1000basepairsofDNAbeingamplified.A2kbsegmentwouldneedanextraminute,whilesegmentsunder200bpdontneedanextensionplateau.Morerecently,PerkinElmerCetushasreportedtheextensionratesforTaq,StoffelandrTthpolymerasesare2-4kbperminute.TaqDNApolymeraseisactiveoverabroadrange,notjustattheextensiontemperature.APerkin-Elmerrepresentativesaidthattheyhavemeasuredelongationratesof75bp/secondat70°C,24bp/secat55°C,and1.5bp/secat35°C.Thus,extensionoccursthroughouttheannealingstep,furtherstABIlizingtheprimer-templateinteractions,butincreasingnonspecificproducts.Afinal5minuteincubationat75#176;Ciscommonlyaddedattheendofthecycles.CloningAmplifiedProducts:TheligationefficiencyofamplifiedDNAvariesfromsequencetosequence.ThecauseoftheproblemiseitheradditionofanAattheendofPCRproducts,and/orTaqpolymeraseenzymeremainingontheendoftheDNAafteramplification.Varioussolutionshavebeendevisedtocompensatefortheseproblems.Perhapsthesimpleststrategywhenyouknowinadvancethatyouaregoingtoclonetheproductsistodesignarestrictionenzymesite(s)tothe5endoftheprimers.Besuretheseareuniquetotheprimersandarenotalsopresentonthesequenceyouareamplifying.Theproductsarethendigestedandsubclonedintoanappropriatelydigestedvector.Notethatsomeenzymesneedacertainnumberofnucleotidesbeyondtheirrecognitionsitefordigestion.In"ImprovedCloningEfficiencyofPolymeraseChainReaction(PCR)ProductsafterProteinaseKDigestion"byCroweetal(1991)Nucl.Acids.Res.19(1):184,theauthorsconcludethatTaqpolymerasestickstotheendsofamplifiedproducts.TheyfoundthatproteinaseKtreatmentincreasedtheyieldoftheirclones.VectorsarecommerciallyavailablethathaveanoverhangingTonthemtocompensateforthepotentialoverhangingAattheendofsomePCRproducts.WehavehadmanysuccessfulligationsofPCRproductsintoblunt-enddigestedvectors,suggestingthatthereisasignificantproportionofmoleculesthatlacktheoverhangingAresidue.Othershavetreatedtheirproductsfor15minuteswith10UofT4DNApolymerase,or30-60minuteswith5-10unitsofKlenowtoproducebluntendproducts.Foranalternatestrategy,seeAslanidisanddeJong,NucleicAcidsResearch18:6069-6074.Themethoddoesnotuseligation,andreportedlyproducesonlyrecombinantswithhighefficiency.SmallinsertscangivelightbluecolonieswhenapUC-basedvectorisemployed.ProductFidelity:;Mutationestimatesvaryfrom10

StockReagent	1x	2x	3x	4x	5x	6x	7x	8x	9x	10x
25mMMgCl2(1.5mMfinal)	1.5	3.45	4.95	6.45	7.95	10.5	12	13.5	15	16.5
10xBuffer(-Mg)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1mMATP (100mMfinal)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1mMGTP(100mMfinal)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1mMTTP(100mMfinal)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1mMCTP(100mMfinal)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
OR--forlabellingDNAsubstituteabovedCTPwith:
1mMdCTP(cold)	2.1	4.83	6.93	9.03	11.13	14.7	16.8	18.9	21	23.1
32P-dCTP(4µCi)	0.4	0.92	1.32	1.72	2.12	2.8	3.2	3.6	4	4.4
----------------------------
Water	0.7	1.61	2.31	3.01	3.71	4.9	5.6	6.3	7	7.7
20µM5primer(1µMfinal)	1.25	2.875	4.125	5.375	6.625	8.75	10	11.25	12.5	13.75
20µM3primer(1µMfinal)	1.25	2.875	4.125	5.375	6.625	8.75	10	11.25	12.5	13.75
TaqPolymerase(5U/µl)(1.5U)	0.3	0.69	0.99	1.29	1.59	2.1	2.4	2.7	3.0	3.3
SUM	17.5	40.25	57.75	75.25	92.75	122.5	140	157.5	175	192.5
DNAtemplate:Q.S.withH20to7.5µl				Dispense17.5µlofmastermixpertube.Add7.5µlofDNAtobringfinalvolumeto25µl.Overlaywith50µlofmineraloil.

-3to10-5andvarywiththelengthoftheproductandthenumberofcycles.Otherparametershavealsobeenfoundthatinfluencethismeasurement.Tomaximizesequencefidelityoftheproducts,orforuseinrandommutagenesis,thefollowingtablemaybehelpful.

Component	IncreasedFidelity	IncreasedInfidelity
dNTP	EqualconcentrationsofdNTPs@40-50µMeach	Unequalconcentrations,1-2mMof3dNTPswith0.1-0.2mMofthefourth
Mg++	1-1.5mM	6-8mMwith0.5mMMn++
Temperature	increase	decrease
[Taq]	decrease	increase
Time	decreaseextensiontime	increaseextensiontime</TD>
#cycles	decrease	increase

ProblemSolvingSection:

(1)Noorfewproductsdetected

Wereallcomponentsadded?

Wasthetemplatedenatured?

Aretheprimersannealingandformingprimerdimers?

Arethecyclingparameterscorrect,i.e.lowenoughforannealingandextension?

(2)Non-specificproductamplification

Aretheprimersspecificforthegeneofinterest?

Wastoomuchpolymeraseadded?

Shouldtheannealingand/orextensiontemperaturesbeincreased?

HastheMg