Severalpointsshouldbementionedconcerningsite-directedmutagenesisusingPCR.First,itisoftendesirabletoreducethenumberofcyclesduringPCRwhenperformingPCR-basedsite-directedmutagenesistopreventclonalexpansionofany(undesired)second-sitemutations.Limitedcyclingwhichwouldresultinreducedproductyield,isoffsetbyincreasingthestartingtemplateconcentration.Second,aselectionmustbeusedtoreducethenumberofparentalmoleculescomingthroughthereaction.Third,inordertouseasinglePCRprimerset,itisdesirabletooptimizethelongPCRmethod.Andfourth,becauseoftheextendaseactivityofsomeThermostablepolymerasesitisoftennecessarytoincorporateanend-polishingstepintotheprocedurepriortoend-to-endligationofthePCR-generatedproductcontainingtheincorporatedmutationsinoneorbothPCRprimers.

Aprotocolisprovidedasafacilemethodforsite-directedmutagenesisandaccomplishestheabovedesiredfeaturesbytheincorporationofthefollowingsteps:(i)increasingtemplateconcentrationapproximately1000-foldoverconventionalPCRconditions;(ii)reducingthenumberofcyclesfrom25-30to5-10;(iii)addingtherestrictionendonucleaseDpnI(recognitiontargetsequence:5-Gm6ATC-3,wheretheAresidueismethylated)toselectagainstparentalDNA(note:DNAisolatedfromalmostallcommonstrainsofE.coliisDam-methylatedatthesequence5-GATC-3);(iv)usingTaqExtenderinthePCRmixforincreasedreliABIlityforPCRto10kb;(v)usingPfuDNApolymerasetopolishtheendsofthePCRproduct,and(vi)efficientintramolecularligationinthepresenceofT4DNAligase.

Protocol.PCR-basedSiteDirectedMutagenesis

• PlasmidtemplateDNA(approximately0.5pmole)isaddedtoaPCRcocktailcontaining,in25ulof1xmutagenesisbuffer:(20mMTrisHCl,pH7.5;8mMMgCl2;40ug/mlBSA);12-20pmoleofeachprimer(oneofwhichmustcontaina5-primephosphate),250uMeachdNTP,2.5UTaqDNApolymerase,2.5UofTaqExtender(Stratagene).
• ThePCRcyclingparametersare1cycleof:4minat94C,2minat50Cand2minat72C;followedby5-10cyclesof1minat94C,2minat54Cand1minat72C(step1).
• TheparentaltemplateDNAandthelinear,mutagenesis-primerincorporatingnewlysynthesizedDNAaretreatedwithDpnI(10U)andPfuDNApolymerase(2.5U).ThisresultsintheDpnIdigestionoftheinvivomethylatedparentaltemplateandhybridDNAandtheremoval,byPfuDNApolymerase,oftheTaqDNApolymerase-extendedbase(s)onthelinearPCRproduct.
• Thereactionisincubatedat37Cfor30minandthentransferredto72Cforanadditional30min(step2).
• Mutagenesisbuffer(1x,115ul,containing0.5mMATP)isaddedtotheDpnI-digested,PfuDNApolymerase-polishedPCRproducts.
• Thesolutionismixedand10ulisremovedtoanewmicrofugetubeandT4DNAligase(2-4U)added.
• Theligationisincubatedforgreaterthan60minat37C(step3).
• ThetreatedsolutionistransformedintocompetentE.coli(step4).

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