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1、1PhysicalPrinciplesandVisual-OMPSoftwareforOptimalPCRDesignJohnSantaLucia,Jr.SummaryThephysicalprinciplesofDNAhybridizationandfoldingaredescribedwithinthecontextofhowtheyareimportantfordesigningoptimalPCRs.Themulti-stateequilibriummodelforcomputingtheconcentration
2、sofcompetingunimolecularandbimolecularspeciesisdescribed.SevenPCRdesign“myths”arestatedexplicitly,andalternativeproperphysicalmodelsforPCRdesignaredescribed.ThischapterprovidesbothatheoreticalframeworkforunderstandingPCRdesignandpracticalguidelinesforusers.TheVisu
3、al-OMP(oligonucleotidemodelingplatform)packagefromDNASoftware,Inc.isalsodescribed.KeyWords:Thermodynamics;nearest-neighbormodel;multi-statemodel;Visual-OMP;secondarystructure;oligonucleotidedesign;software.1.IntroductionSingle-targetPCRisgenerallyregardedasarobust
4、andreliabletechniqueforamplifyingnucleicacids.ThisreputationiswelldeservedandisaresultoftheinherentnatureofPCRtechnology,thecreativityofawidevarietyofscientistsandengineers,andthehugefinancialinvestmentofprivateindustryaswellasgovernmentfunding.Anincompletelistofs
5、omeoftheimportantinnovationsincludesavarietyofengineeredthermostablepolymerases,well-engineeredthermocyclinginstruments,hot-startPCR,exonuclease-deficientpolymerases,additionofdimethylsulfoxide(DMSO),bufferoptimization,aerosol-blockingpipettetips,anduseofuracilDNA
6、glycosylasetominimizecontaminationartifacts.Despitetheseinnovationsandthelargeinvestment,therearemanyFrom:MethodsinMolecularBiology,vol.402:PCRPrimerDesignEditedby:A.Yuryev©HumanaPress,Totowa,NJ34SantaLuciaaspectsofPCRthatarestillnotwellunderstood(suchasthedetaile
7、dkinetictimecourseofreactionsthatoccurduringthermocycling).Thesegapsinourknowledgeresultinless-than-perfectdesignsoftware;thehumanexpertsarenotperfecteither.Nonetheless,thereisaseriesofwidelybelievedmythsaboutPCRthatresultinpoordesigns.Thischapterisdevotedtostatin
8、gexplicitlysomeofthesemythsandprovidingexplanationsandguidelinesforimprovedPCRdesign.TheseprinciplesarefullyimplementedinthecommercialpackagefromDNASoft
