11Chapter9

PCRandSite-directedmutagenesis223Introduction2.PCR3.Site-directedmutagenesis35513October1993TheRoyalSwedishAcademyofScienceshasdecidedtoawardthe1993NobelPrizeinChemistryforcontributionstothedevelopmentofmethodswithinDNA-basedchemistry,

withhalfto

DrKaryB.Mullis,LaJolla,California,U.S.A.,forhisinventionofthepolymerasechainreaction(PCR)method,

andhalfto

ProfessorMichaelSmith,UniversityofBritishColumbia,Vancouver,Canada,forhisfundamentalcontributionstotheestablishmentofoligonucleotide-based,site-directedmutagenesisanditsdevelopmentforproteinstudies.

DrKaryB.MullisProfMichaelSmithPressRelease:The1993NobelPrizeinChemistry6

Polymerasechainreaction:itderivesitsnamefromoneofitskeycomponents--aDNApolymeraseusedtoamplifyapieceofDNAbyinvitro

enzymaticreplication.PCRisalsocallednon-cellmolecularclonesystem.

ThebasiccomponentsofPCRinclude:DNAtemplate,primers(whicharecomplementarytotheDNAregionsat5’-primeror3’-primerendsoftheDNA),deoxynucleosidetriphosphates(dATP,dCTP,dGTP,dTTP),andTaqDNA

polymeraseorotherDNApolymerase.6PCR(polymerasechainreaction)7--thereplicationprocessofDNAinvivobecomeasimpleprocessinvitro--generatemillionscopiesofthetargetDNAfragmentswithoutclone7PCR8Site-directedmutagenesis

Site-directedmutagenesisofgeneisamolecularbiologytechniqueinwhichamutationiscreatedatadefinedsiteinaDNAmolecule,usuallyacircularmolecularknownasaplasmid.Themutationtypesinclude: multiplepointmutations

insertionmutagenesis

deletionmutagenesisabasictechniqueofreversegeneticsatoolofproteinengineeringacommongene-manipulationtechniqueinlabs810(1)PCR

1)principle

2)theramalcyclingprofileforstandardPCR3)basicreactionandthefunctionofeverycomponents4)theidentifyofPCRproduct5)optimizedconditionsofPCR(2)VariationsonthebasicPCRtechnique

1)RT-PCR

2)NestedPCR 3)insituPCR 4)MultiplexPCR 5)Real-timequantitativePCR

1012Semi-conservationreplication141)Principle:semi-conservativereplicationofDNA

1515162)ThermalCyclingProfileforStandardPCRInitialDenaturation:Thisstepconsistsofheatingthereactiontoatemperatureof94-95℃whichisheldof1-9minutes.Eachcycleincludesthreesuccessivesteps:

Denaturation:at94-96℃,duringwhichtheDNAisdenaturedintosinglestrands.

Annealing:at50-65℃,duringwhichtheprimershybridizeor“anneal”totheircomplementarysequencesoneithersideofthetargetsequence.

Extensive:72℃,duringwhichthepolymerasebindsandextendsacomplementaryDNAstrandfromeachprimerandaddapproximately60basespersecondat72℃.Postextensionandholding:cyclingshouldconcludewithafinalextesionat72℃for5-15minutestopromotecompletionofpartialextensionproductsandthenholdingat4℃.17PCRCyclesReviewDenaturalization:94-95℃,30secPrimerAnnealing:50-65℃,45secExtensionofDNA:72℃,

(DNAsynethesistimedependsonproductsize)NumberofCycles:25-3595℃30s50-65℃30s72℃?min95℃5min25-35

cycles72℃7min4℃forever18TheoreticalPCRamplificationofatargetfragmentwithincreasingnumberofcyclesTheextensionreactioncreatetwodouble-strandedtargetregions,eachofwhichcanagainbethetemplateforasecondcycleofhybridizationandextension.Intheory,byrepeatedcyclesofheatdenaturation,primerhybridizationandextention,therefollowsarapidexponentialaccumulationoftargetDNA.Theaccumulationisnotstrictlyadoublingateachcycleintheearlyphase.At30cyclesthereare～1×109copies.1billionmoleculesfromtheoriginalonestartedwith.182021TheamplificationefficiencyofpracticalPCR21

Inpractice,

theamplificationefficiencyofPCRislowerthanthetheory.DifferenttemplateDNAhavedifferentamplificationefficiency.Afternumberofcycles,thereactionslowsastheDNApolymeraselosesactivityandasconsumptionofreagentssuchasdNTPsandprimerscausesthemtobecomelimiting.Thencometothe“Plateau”,inwhichphasenomoreproductaccumulatesduetotheexhaustionofreagentsandenzyme.

233)Basicreactionandthefunctionofeverycomponents2324PCRReactionComponents24

Template:previouslyisolatedandpurified.

Twoprimers:toflankthetargetsequence.

Fourdeoxynucleosidestriphosphate(dNTPs):toprovideenergyandnucleosidesforthesynthesisofDNA.

Buffersystem:containingmagnesium.

DNApolymerase26Template1μghumansingle-copygenomicDNA3×105targets10ngyeastDNA3×105

1ngE.coliDNA3×105

1ng1kbDNA9×106

1%M13plaque106

500ngmaximunamounthumangenomicDNA1-10ngbacterialDNA0.1-1ngplasmidDNA27PCRPrimersPrimersrangefrom15to30nucleotides,aresingle-stranded,andareusedforthecomplementarybuildingblocksofthetargetsequence.AprimerforeachtargetsequenceontheendofyourDNAisneeded.Thisallowsbothstrandstobecopiedsimultaneouslyinbothdirections.2830TmAnnealingtemperature:Tm-5(℃)primelength:20nucleotidesG+C=10A+T=10(G+C)%=50a.Tm=(A+T)×2+(G+C)×4Tm=10×2+10×4=60℃b.Tm=62.3+0.41

(G+C)%-500/N(N:thelengthofprime)Tm=62.3+0.41×50-500/20=57.8℃3132Buffersolution

Functionsofbuffersolution:a.MaintainthepHrangefortheTaq:Tris-HCl(pH8.3-8.8,20℃)

b.Bivalentcation:Mg2+(MgCl2,MgSO4),Mn2+c.Primeannealing:KCld.Protectenzymeactivity:

gelatin,BSA,Tween-20orDTT3233Mg2+

Mg2+isanessentialcomponentoftheTaqDNApolymerase

TheconcentrationofMg2+affectsenzymespecificity,reactionyield,Tm,formationofprimerdimer...(DNAtemplate,primers,dNTPscanbindwithMg2+,anddecreasetheconcentrationofMg2+）theTaqDNApolymeraseneeddisassociatedMg2+,sothequantityofMg2+inPCRshouldhigherthantheconcentrationofdNTPsby0.2-2.5mmol/L.3334dNTPs

AbasicmaterialofDNAreplication.thestandardconcentrationis20~200mol/LTheconcentrationofthefourkindsofdNTPshouldequal.toolow:slowdownthereactionspeedtoohigh:increasetheformationofnonspecificproductsandwhentheconcentrationofdNTPhigherthan50mM,theactivityofTaqDNApolymerasewillbeinhibited.3435DNApolymerasea.KlenowDNApolymeraseb.TaqDNApolymerasec.High-fidelityDNApolymeraseProfreadingornotHigherconcentration:non-specialcopyLowerconcentartion:non-enoughproduct3536KeyFeaturesFidelityofincorporationProcessivity,rateofsynthesisStability,half-lifeatdifferenttemperatures37TaqDNApolymeraseisseparatefromtothethermophilicbacterium(thermusaquatious).Half-time:92.5℃＞2h95℃40min97.5℃5minTaqDNApolymeraselacksthe3’→5’proof-readingactivitycommonlypresentinotherpolymeraseTaqDNApolymerasemis-incorporates1basein104A400bptargetwillcontainanerrorin33%ofmoleculesafter20cyclesErrordistributionwillberandomUseaproof-readingthermo-stableenzymeratherthanTaqTaqDNApolymerase

38TVector3940High-fidelityDNApolymeraseVentDNApolymerase(NewEnglandBlabs)Half-life:1.8hat100℃（MgSO4）

5minTaqTliDNApolymerase(Promega)PwoDNApolymerase(BM-Roche)Half-life:＞2hat100℃KODPlusDNApolymerase(ToYoBo):blunt-endedPfuDNApolymerase(Stratagene):blunt-ended41PCRRequirementsMagnesiumchloride:5-2.5mMBuffer:pH8.3-8.8dNTPs:20-200μMPrimers:0.1-0.5μMDNAPolymerase:1-2.5units(U)TargetDNA:1μg424)methodsofproductdetectionandanalysisGelelectrophoresisHybridizationPCR-ELISA4243Productdetection43445)optimizedconditionsofPCR

primers：specificationAnnealingtemperature： TouchdownandstepdownThequalityandquantityoftemplate(avoidcontamination)4445LargefragmentamplificationOptimisingprimers46Optimisingannealingtmperature4747

TheplanofalabPCRreagentsprepareregion

PCRtemplateregionMixregionPCRamplificationanddetectionregion482.

BriefintroductionofvariantsofPCR

48491)RT-PCR

RNAastemplateTherearetwostepsinRT-PCR:a.Reversetranscription:mRNA→cDNA

reversetranscriptase:RNAdependedDNApolymerase fromM-MLV,AMVreverseprimers: genespecificprimer Oligo(dT)(12-18bases) randomhexamer

b.PCR49502)nestedPCRIncreasesthespecificityofDNAamplification,byreducingbackgroundduetonon-specificamplificationofDNA.TwosetsofprimersarebeingusedintwosuccessivePCRs.Therelationbetweenprimersisillustratedasfollow:Inthefirstreaction,primer1andprimer4areusedtogenerateDNAproducts,whichbesidestheintendstarget.TheproductsmentionedabovearethenusedinasecondPCRwithprimer2andprimer3whosebindingsitesarecompletelyorpartiallydifferentfromandlocated3’ofeachoftheprimersusedinthefirstreaction.Iftheproductoffirstreactioncanbeseeninthegel,thenuse1/104-105ofitasthetemplateinthesecondreaction.Advantages:moresuccessfulinspecificallyamplifyinglongDNAfragments thanconventional513)insituPCRInsituPCRisusedintheamplificationofspecificDNAorRNAintissuecells.Theproductcanbedetecteddirectlyoranalysisbyinsituhybridization.Thismethodcanbeusedtovirusdetection,tumorformation,analysisofRNAtransportation…5152524)multiplexPCR

Usemultiple,uniqueprimersetswithinasinglePCRmixturetoproduceamplicationsofvaryingsizesspecifictodifferentDNAsequences.Thismethodcanbeusedto

pathogendetection,geneticillnessdiagnose…5353RealtimePCRisanewtechniqueinventedin1996andhavedevelopeduntilnow.

Themeaningof“realtime”:?

QuantitativelymeasurementandanalysiscompanywiththePCR?AmplificationoftargetDNAcompanywiththeproductanalysis-thismethodusefluorescentdyes,suchasSybrGreen,or

fluorophore-containingDNAprobes,suchasTaqMan,tomeasuretheamountofamplifiedproductinthesametube,bythesameequipment.5)RealtimePCR54MethodsofquantitativelymeasureFluorescentdyesandfluorophore-containingDNAprobes Fluorescentdyesiseasytohandle Fluorophore-containingDNAprobeshavemorespecification5455Fluorescentdye：SYBRGreenImethod5556(A)mexE(Primers1)

(B)mexE(primers2)MeltCurveChart(FvsT)MeltCurvePeakChart(-dF/dtvsT)MeltCurveChart(FvsT)MeltCurvePeakChart(-dF/dtvsT)MeltCurve5757Ct：CmeansCycle,tmeansthreshold,

TherelationbetweenCtandoriginalcopyoftemplateisreverseratioThesiteofthreshold:

threshold=10’SDcycle3-15

58QuantificationprincipleTherelationbetweenCtandoriginalcopyoftemplateisreverseratio.5859AdvantagesofReal-timequantitativefluorescent

PCRa.quantitativefluorescentPCRtakeinacompletelyclosed statement,andavoidfakeresultefficiently.b.fluorescentprobehasmoresensitivityandspecification.The quantityoftemplatecanbeidentifiedmoreaccurately.c.thereisnoelectrophoresisafterPCR,cansavetimeandeasyto

handle.5960TaqManprobea.TaqManprobecontaina“reporterdye”anda“quencher”,therearenofluorescesbeforePCR.b.Whenanneal,bothprimerandprobebindwithtemplateFluorescesproducebythe“reporterdye”whenthe"quencher"isremovedfromthefragmentduringthePCRextensioncycle.Theintensityoffluorescesisproportionatewiththequantityoftemplate.

6061623.Site-directedmutagenesisFunction:Itisveryusefultobeabletochangejustone,orafewspecificnucleotidesinasequencetotestahypothesis.

Methods:

(1)Thesingle-primermethod (2)Cassettemutagenesis (3)ThePCRmethodofmutagenesis6263Pro