Primer Design Guide for PCR - PREMIER Biosoft

3. Tm of Product: Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. Togglenavigation HOME COMPANY PRODUCTS ORDERING SUPPORT BLOG References>>PCRPrimer PCR PrimerDesignGuidelines PCR (PolymeraseChainReaction) PolymeraseChainReaction iswidelyheldasoneofthemostimportant inventionsofthe20thcenturyinmolecular biology.Smallamountsofthegenetic materialcannowbeamplifiedtobeable toaidentify,manipulateDNA,detect infectiousorganisms,includingtheviruses thatcauseAIDS,hepatitis,tuberculosis, detectgeneticvariations,includingmutations, inhumangenesandnumerousothertasks. PCRinvolvesthefollowingthreesteps:Denaturation,AnnealingandExtension.First, thegeneticmaterialisdenatured,converting thedoublestrandedDNAmoleculesto singlestrands.Theprimersarethen annealedtothecomplementaryregions ofthesinglestrandedmolecules.In thethirdstep,theyareextendedby theactionoftheDNApolymerase.All thesestepsaretemperaturesensitive andthecommonchoiceoftemperatures is94oC,60oC and70oCrespectively.Good primerdesignisessentialforsuccessful reactions.Theimportantdesignconsiderations describedbelowareakeytospecific amplificationwithhighyield.Thepreferred valuesindicatedarebuiltintoall ourproductsbydefault. 1.PrimerLength:Itisgenerallyacceptedthattheoptimal lengthofPCRprimersis18-22bp.This lengthislongenoughforadequatespecificity andshortenoughforprimerstobind easilytothetemplateattheannealing temperature. 2.PrimerMeltingTemperature:PrimerMeltingTemperature(Tm) bydefinitionisthetemperatureat whichonehalfoftheDNAduplexwill dissociatetobecomesinglestranded andindicatestheduplexstability. Primerswithmeltingtemperaturesin therangeof52-58oCgenerally producethebestresults.Primerswith meltingtemperaturesabove65oC haveatendencyforsecondaryannealing. TheGCcontentofthesequencegives afairindicationoftheprimerTm. Allourproductscalculateitusing thenearestneighborthermodynamictheory, acceptedasamuchsuperiormethodfor estimatingit,whichisconsideredthemost recentandbestavailable. Formulafor primerTmcalculation: MeltingTemperatureTm(K)={ΔH/ ΔS+Rln(C)},OrMeltingTemperature Tm(oC)={ΔH/ ΔS+Rln(C)}-273.15where ΔH (kcal/mole):HistheEnthalpy. Enthalpyistheamountofheatenergy possessedbysubstances.ΔHis thechangeinEnthalpy.Intheabove formulatheΔHisobtainedby addingupallthedi-nucleotidepairs enthalpyvaluesofeachnearestneighbor basepair. ΔS (kcal/mole):Sistheamountof disorderasystemexhibitsiscalled entropy.ΔSischangeinEntropy. Hereitisobtainedbyaddingupall thedi-nucleotidepairsentropyvalues ofeachnearestneighborbasepair.Anadditionalsaltcorrection isaddedastheNearestNeighborparameters wereobtainedfromDNAmeltingstudies conductedin1MNa+bufferandthis isthedefaultconditionusedforall calculations. ΔS (saltcorrection)=ΔS(1MNaCl )+0.368xNxln([Na+]) Where Nisthenumberofnucleotidepairs intheprimer(primerlength-1). [Na+]issaltequivalentinmM. [Na+]calculation: [Na+]= Monovalentionconcentration+4xfree Mg2+. 3.PrimerAnnealingTemperature:Theprimermeltingtemperature istheestimateoftheDNA-DNAhybrid stabilityandcriticalindetermining theannealingtemperature.Toohigh Tawillproduceinsufficient primer-templatehybridizationresulting inlowPCRproductyield.ToolowTamaypossiblyleadtonon-specificproducts causedbyahighnumberofbasepair mismatches,.Mismatchtoleranceisfound tohavethestrongestinfluenceonPCR specificity. Ta=0.3xTm(primer)+0.7 Tm(product)–14.9 where, Tm(primer)=MeltingTemperature oftheprimers Tm(product) =Meltingtemperatureoftheproduct 4.GCContent:The GCcontent(thenumberofG'sandC's intheprimerasapercentageofthe totalbases)ofprimershouldbe40-60%. 5.GCClamp:The presenceofGorCbaseswithinthe lastfivebasesfromthe3'endofprimers (GCclamp)helpspromotespecificbinding atthe3'endduetothestrongerbonding ofGandCbases.Morethan3G'sor C'sshouldbeavoidedinthelast5 basesatthe3'endoftheprimer. 6.PrimerSecondaryStructures:Presenceoftheprimersecondarystructures producedbyintermolecularorintramolecular interactionscanleadtopoororno yieldoftheproduct.Theyadversely affectprimertemplateannealingand thustheamplification.Theygreatly reducetheavailabilityofprimersto thereaction. i) Hairpins:Itisformedby intramolecularinteractionwithinthe primerandshouldbeavoided.Optimally a3'endhairpinwithaΔGof -2kcal/molandaninternalhairpin withaΔGof-3kcal/molistolerated generally. ΔG definition:TheGibbsFreeEnergy Gisthemeasureoftheamountofwork thatcanbeextractedfromaprocess operatingataconstantpressure.It isthemeasureofthespontaneityof thereaction.Thestabilityofhairpin iscommonlyrepresentedbyitsΔG value,theenergyrequiredtobreak thesecondarystructure.Largernegative valueforΔGindicatesstable, undesirablehairpins.Presenceofhairpins atthe3'endmostadverselyaffects thereaction. ΔG =ΔH–TΔS ii)SelfDimer: Aprimerself-dimerisformedbyintermolecularinteractions betweenthetwo(samesense)primers, wheretheprimerishomologoustoitself. Generallyalargeamountofprimersare usedinPCRcomparedtotheamountof targetgene.Whenprimersformintermolecular dimersmuchmorereadilythanhybridizing totargetDNA,theyreducetheproduct yield.Optimallya3'endselfdimerwith aΔGof-5kcal/molandaninternal selfdimerwithaΔGof-6kcal/mol istoleratedgenerally. iii)Cross Dimer:Primercrossdimersareformed byintermolecularinteractionbetween senseandantisenseprimers,wherethey arehomologous.Optimallya3'endcross dimerwithaΔGof-5kcal/moland aninternalcrossdimerwithaΔG of-6kcal/molistoleratedgenerally. 7.Repeats:Arepeat isadi-nucleotideoccurringmanytimes consecutivelyandshouldbeavoided becausetheycanmisprime.Forexample: ATATATAT.Amaximumnumberofdi-nucleotide repeatsacceptableinanoligois4di-nucleotides. 8.Runs:Primers withlongrunsofasinglebaseshould generallybeavoidedastheycanmisprime. Forexample,AGCGGGGGATGGGGhasruns ofbase'G'ofvalue5and4.Amaximum numberofrunsacceptedis4bp. 9.3'EndStability:ItisthemaximumΔGvalueof thefivebasesfromthe3'end.Anunstable 3'end(lessnegativeΔG)will resultinlessfalsepriming. 10.AvoidTemplateSecondary Structure:A singlestrandedNucleic acidsequencesishighlyunstableand foldintoconformations(secondarystructures). Thestabilityofthesetemplatesecondary structuresdependslargelyontheir freeenergyandmeltingtemperatures(Tm). Considerationoftemplatesecondary structuresisimportantindesigning primers,especiallyinqPCR.Ifprimers aredesignedonasecondarystructures whichisstableevenabovetheannealing temperatures,theprimersareunable tobindtothetemplateandtheyield ofPCRproductissignificantlyaffected. Hence,itisimportanttodesignprimers intheregionsofthetemplatesthat donotformstablesecondarystructures duringthePCRreaction.Ourproducts determinethesecondarystructuresof thetemplateanddesignprimersavoidingthem. 11.AvoidCrossHomology:To improvespecificityoftheprimersit isnecessarytoavoidregionsofhomology.Primersdesignedfor asequencemustnotamplifyothergenes inthemixture.Commonly,primersare designedandthenBLASTedtotestthe specificity.Ourproductsofferabetter alternative.Youcanavoidregionsof crosshomologywhiledesigningprimers. YoucanBLASTthetemplatesagainst theappropriatenon-redundantdatabase andthesoftwarewillinterpretthe results.Itwillidentifyregionssignificant crosshomologiesineachtemplateand avoidthemduringprimersearch. Parameters forPrimerPairDesign 1.AmpliconLength:Theampliconlengthisdictatedbythe experimentalgoals.ForqPCR,thetarget lengthiscloserto100bpandforstandard PCR,itisnear500bp.Ifyouknow thepositionsofeachprimerwithrespect tothetemplate,theproductiscalculated as:Productlength=(Positionofantisense primer-Positionofsenseprimer)+1. 2.ProductPosition:Primercanbelocatednearthe5'end, the3'endoranywherewithinspecified length.Generally,thesequenceclose tothe3'endisknownwithgreater confidenceandhencepreferredmost frequently. 3.TmofProduct:MeltingTemperature(Tm) isthetemperatureatwhichonehalf oftheDNAduplexwilldissociateand becomesinglestranded.Thestability oftheprimer-templateDNAduplexcan bemeasuredbythemeltingtemperature (Tm). 4.OptimumAnnealingTemperature (TaOpt):Theformula ofRychlikismostrespected.Ourproducts usethisformulatocalculateitand thousandsofourcustomershavereported goodresultsusingitfortheannealing stepofthePCRcycle.Itusuallyresults ingoodPCRproductyieldwithminimum falseproductproduction. TaOpt=0.3x(Tmofprimer) +0.7x(Tmofproduct)- 14.9 where Tmofprimeristhemelting temperatureofthelessstableprimer-template pair Tmofproductisthemelting temperatureofthePCRproduct. 5.PrimerPairTmMismatch Calculation:Thetwoprimers ofaprimerpairshouldhaveclosely matchedmeltingtemperaturesformaximizing PCRproductyield.Thedifferenceof 5oCormorecanleadnoamplification. PrimerDesignusingSoftware AnumberofprimerdesigntoolsareavailablethatcanassistinPCRprimerdesignfornewandexperiencedusersalike.Thesetoolsmayreducethecostandtimeinvolvedinexperimentationbyloweringthechancesoffailedexperimentation. PrimerPremierfollowsalltheguidelinesspecifiedforPCRprimerdesign.PrimerPremiercanbeusedtodesignprimersforsingletemplates,alignments,degenerateprimerdesign,restrictionenzymeanalysis.contiganalysisanddesignofsequencingprimers. TheguidelinesforqPCRprimerdesignvaryslightly.SoftwaresuchasAlleleIDandBeaconDesignercandesignprimersandoligonucleotideprobesforcomplexdetectionassayssuchasmultiplexassays,crossspeciesprimerdesign,speciesspecificprimerdesignandprimerdesigntoreducethecostofexperimentation. PrimerPlexisasoftwarethatcandesignprimersforMultiplexPCRandmultiplexSNPgenotypingassays.   QUICKLINKS News|Events Testimonials Citations&Reviews Sitemap EXPLORE OurSoftware TryNow FreeEducationalLicense FreeTools ORDERING PriceList GenerateaQuote OrderNow RequestaW-9Form SUPPORT ContactUs BookaMeeting FAQ Suggestions ©1994-PREMIERBiosoft.Allrightsreserved. customerservicesoftwaretechnicalsupport LiveChatbyComm100