DesigningPCRprograms TherequirementofanoptimalPCRreactionistoamplifyaspecificlocuswithoutanyunspecificby-products.Therefore,annealingneedstotakeplaceatasufficientlyhightemperaturetoallowonlytheperfectDNA-DNAmatchestooccurinthereaction.Foranygivenprimerpair,thePCRprogramcanbeselectedbasedonthecomposition(GCcontent)oftheprimersandthelengthoftheexpectedPCRproduct.Inthemajorityofthecases,productsexpectedtobeamplifiedarerelativelysmall(from0.1to2-3kb).(Forlong-rangePCR(amplifyingproductsof10to20-30kb)commercialkitsareavailable).TheactivityoftheTaqpolymeraseisabout2000nucleotides/minuteatoptimaltemperature(72-78oC)andtheextensiontimeinthereactioncanbecalculatedaccordingly. 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. 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.
BasicPrinciples(seealsoPage01)
Influenceofannealingtemperatureandnumberoflociamplified
Numberofcycles
