DesigningPCRprograms

BasicPrinciples(seealsoPage01)

TherequirementofanoptimalPCRreactionistoamplifyaspecificlocuswithoutanyunspecificby-products.Therefore,annealingneedstotakeplaceatasufficientlyhightemperaturetoallowonlytheperfectDNA-DNAmatchestooccurinthereaction.Foranygivenprimerpair,thePCRprogramcanbeselectedbasedonthecomposition(GCcontent)oftheprimersandthelengthoftheexpectedPCRproduct.Inthemajorityofthecases,productsexpectedtobeamplifiedarerelativelysmall(from0.1to2-3kb).(Forlong-rangePCR(amplifyingproductsof10to20-30kb)commercialkitsareavailable).TheactivityoftheTaqpolymeraseisabout2000nucleotides/minuteatoptimaltemperature(72-78oC)andtheextensiontimeinthereactioncanbecalculatedaccordingly.

  • Astheactivityoftheenzymemaynotbealwaysoptimalduringthereaction,aneasyruleappliedsuccessfullybytheauthorwastoconsideranextensiontime(inminutes)equaltothenumberofkboftheproducttobeamplified(1minfora1kbproduct,2minforatwokbproductetc.).Lateron,aftertheproduct(s)become\"known\",extensiontimemaybefurtherreduced.
  • Manyresearchersusea2-5minutesfirstdenaturingstepbeforetheactualcyclingstarts.ThisissupposedtohelpdenaturingthetargetDNAbetter(especiallythehardtodenaturetemplates).Also,afinallastextensiontime,of5-10minutes,isdescribedinmanypapers(supposedlytohelpfinishtheelongationofmanyormostPCRproductsinitiatedduringthelastcycle).Boththesestepshavebeentestedforanumerofdifferentloci,and,basedonthisexperience,neitherthefirstdenaturingnorthelastextensiontimechangedinanywaytheoutcomeofthePCRreaction.Therefore,itistheauthor\"shABItnottousethesesteps(lightblueinthetablebelow)anymore.
  • Theannealingtimecanbechosenbasedonthemeltingtemperatureoftheprimers(whichcanbecalculatedusingothemanyapplications,freelyavailableformolecularBIOLOGists).Thismaywork,butsometimestheresultsmaynotmatchtheexpectations.Therefore,asimpleprocedureusedmanytimesbytheauthorwastouseaninitialannealingtemperatureof54oC(usuallygoodformostprimerswithalengtharound20bpormore).Ifunspecificproductsresult,thistemperatureshoudbeinccreased.Ifthereactionisspecific(onlytheexpectedproductissynthesized)thetemperaturecanbeusedasis.
  • Fortheseventyorsoprimersusedduringthiswork,adenaturingtimeof30-60secondswassufficienttoachievegoodPCRproducts.Tolongadenaturingtime,willincreasethetimetheTaqpolymeraseissubjectedathightemperatures,andincreasesthepercentageofpolymerasemoleculesthatlosetheiractivity.
  • Numberofcycles.Ingeneral,30cyclesshouldbesufficientforausualPCRreaction.Anincreasednumberofcycleswillnotdramaticallychangetheamountofproduct(seebelow).
    • Influenceofannealingtemperatureandnumberoflociamplified

    LikeanyotherPCR,multiplexreactionsshouldbedoneatastringentenoughtemperature,allowingamplificationofalllociofinterestwithout\"background\"by-products.Althoughmanyindividuallocicanbespecificallyamplifiedatanannealingtemperatureof56°-60°C,experimentsshowedthatloweringtheannealingtemperatureby4-6°Cwasrequiredforthesamelocitobeco-amplifiedinmultiplexmixtures.ThisisdemonstratedinFig.19below,showingthesamePCRreactionsperformedinconditionsinwhichtheonlyparameterchangedwastheannealingtemperature.ForthemultiplexaPCRamplificationofmixturesCandC*,anannealingtemperatureof54°Cseemsthemostappropriate,althoughtheindividualloci(forexample\"Y\")couldbeamplifiedat60°C.At54°C,althoughsomeunspecificamplificationprobablystilloccursinthemultiplexreaction,itisovercomebytheconcurrentamplificationofanincreasednumberofspecificlociandthusremainsinvisIBLe.

    InPCR,duetodifferencesinbasecomposition,lengthofproductorsecondarystructuresomelociaremoreefficientlyamplifiedthanothersWhenmanylociaresimultaneouslyamplified(multiplexed),themoreefficientlyamplifiedlociwillnegativelyinfluencetheyieldofproductfromthelessefficientloci.ThisphenomenonisdueinparttothelimitedsupplyofenzymeandnucleotidesinthePCRreaction.Therefore,inthemultiplexprocedurethemoreefficientlyamplifiedlocicompetebetterandtakeoverthelessefficientlyamplifiedproducts,thusrenderingthemlessvisibleorinvisible.

    (Figure19below,depictsacomplexsituationinwhichannealingtemperature,numberofsimultaneouslyamplifiedlociandbufferconcentrationwerechangedinparallelreactions).

Fig.19.MultiplexamplificationofmixtureC*(firstthreelanesineachgel),primerpair\"Y\"(lanes4to6,bluearrows)andmixtureC(lanes7to12in1xor2xPCRbuffer)onthreedifferenttemplateDNAsusingthreePCRprogramsdifferinginannealingtemperature(48°C,54°Cor59°C).Lanes1-9oneachgelshowreactionsin1xPCRbuffer.Lanes10-12oneachgelshowreactionsin2xPCRbuffer.Lanes7-12oneachgel(under\"1x\"and\"2x\")werewithprimermixtureC.TheunmarkedlanesaretheMarker(1kbladder).ThefivearrowstotheleftsideofthefirstgelindicatetheexpectedproductsofmixC*(fiveproducts).Thelongestspecificproductoneachgelismarkedbyaredarrow.Magentaarrowindicatesastrongunspecificproduct.YellowarrowsindicatethetwoextraproductsexpectedinmixC(totalofsevenproducts)comparedwithC*.BluearrowsindicatepositionofproductY(eitherbyitselforinthemultiplexmixture)inthefirstgelorthelackofproductYinsomeofthereactionsfromthelasttwogels.Multiplexamplificationat48°Cshowsmanyunspecificbands.In1xPCRbuffer,theYproductisstrongerwhenamplifiedinmixtureC*(5primerpairs)thaninmixtureC(7primerpairs)showingthat,atleastforsomeproducts,anincreasednumberofsimultaneouslyamplifiedlocicaninfluencetheyieldofsomeindividualloci.RaisingthePCRbufferconcentrationfrom1xto2xallowsamoreevenamplificationofallspecificproductsandhelpsindecreasetheintensityofmanylongerunspecificproducts(comparelanes7-9vs.10-12).Thestrong470-480bpunspecificband(magentaarrow)seenwith2xbufferwaseliminatedbyvaryingtheproportionofdifferentprimersinthereaction(comparewithCinFig1).At59°CtheYproductcanbeseenonlywhen2xbufferisusedorwhenthelocusisamplifiedalone.

Numberofcycles

PrimermixC*wasusedtoamplifytwodifferentgenomicDNAtemplates,stoppingthereactionafterincreasingnumbersofcycles(Fig.20).ForthesameDNAtemplate,resultswerereproducibleamongallvialsalthoughoneofthetwogenomicDNAswasbetter,probablyduetothehigherqualityand/oramountofDNA.Themostobviousvariationintheamountofproductswasaround24cycles(forethidiumbromidestainedgels).28-30cyclesareusuallysufficientinareaction.Littleornoquantitativechanges(i.e.,relativeamountsofPCRproducts)wereobservedwithincreasingcyclenumberup45.Littlequantitativegainwasnoticedwhenincreasingthenumberofcyclesupto60(Fig.21)

Fig.20.MultiplexamplificationofmixtureC*usingtwodifferentDNAtemplatesandincreasingthenumbersofcyclesbyunitsofthree.

Fig.21.MultiplexamplificationofmixtureC*usingtthesamePCRprogramandincreasingthenumberofcyclesbyunitsoften(upto60).Noadditionalingredientswereaddedinthereactions.