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KaryMullis1983,POLYMERASECHAINREACTION,AlicencetodomolecularbiologyAkeycentraltechniquethathasrevolutionisedmolecularandconsequentlycellbiology,InthisLecture,PrincipleofPCRPCR,thebasicsOptimizethePCRandtroubleshootApplicationofPCRReverse-TranscriptionPCR（反轉(zhuǎn)錄PCR〕PCRcloning（PCR克隆技術(shù)〕Real-TimePCR（實時定量PCR〕HistoryofPCR,,PolymeraseChainReaction,PCR是一種體外酶促合成特定DNA片斷的技術(shù)，是根據(jù)人類的需要對復(fù)雜生命過程的一種簡單化的模擬。PCR技術(shù)的原理是DNA半保留復(fù)制。,DNAReplicationinvivo,http://www.accessexcellence.org/AB/GG/collaboration.html,StrandseparationSynthesisofshortRNAprimersSynthesisofnewDNAstrands,,DENATURATION93C-95C,DNAreplicationinvitro,,ThePCRProcessinvitro,,TemplateDNA:ThesampletoamplifiedPrimersShort,specificsegementsofDNA,provideSPECIFICITYdATP,dTTP,dCTP,dGTPThermostableDNApolymerase(e.g.,Taq,Pfu)Bufferandsalts(KCl,MgCl2)Optional:BSA,DMSO,Formamide,TYPICALREACTIONMIXTURE,25or50?lsinamicroEppendorf(0.5ml)tube,(1.0-4.5mM),ThePCRinvitro,,http://vector.cshl.org/resources/BiologyAnimationLibrary.htm,,,,,PCR,Agarosegelelectrophoresis,Thefinalproduct,UVvisualisation,3-4hours,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCR–THEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCR–THEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCR–THEREACTIONCOMPONENTS,ThermastatDNApolymerase:Taq,Pfu,Vente.g.,Isolatedfromathermophilicbacterium(Thermusaquaticus)fromaculturederivedfromahotspringinYellowstone.CatalyzesDNApolymerizationatelevatedtemperature(72C)andisresistantto98C.Similarenzymeshavehavebeenidentifiedfromotherthermophilicbacteria,suchasthoselivingindeepseavents.,,NumberofoptionsavailableTaqpolymerasePfupolymeraseTthpolymeraseHowbigistheproduct?100bp40-50kbWhatisendpurposeofPCR?Sequencing/mutationdetection-NeedhighfidelitypolymeraseCloning(TAcloning)-TaqDNAPolymerase,,CHOOSEYOURPOLYMERASEWITHCARE,,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCR–THEREACTIONCOMPONENTS,Length~18-30nt(21nt)Basecomposition;50-60%GCrichpairsshouldhaveequivalentTmsTm=[(numberofA+Tresidues)x2C]+[(numberofG+Cresidues)x4C]InitialuseTm–5CAvoidinternalhairpinstructuresnosecondarystructureAvoidaTatthe3’endAvoidoverlapping3’ends–willformprimerdimersCanmodify5’endstoaddrestrictionsitesetc,PRIMERDESIGNISVITAL,optimiseforeachsetofprimers,OPTIMISEPCRCONDITIONS--Primer,X,?,DNAprimersforPCR,,PRIMERDESIGN,Primer5fromPremierprimerCo.,Alsoavailableoninternethttp://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.htmlOligoSys:,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCR–THEREACTIONCOMPONENTS,TITRATEYOURMg2+CONCENTRATION!,43.532.521.51mM,Normally,1.5mMMgCl2isoptimalBestsuppliedasseparatetubeAlwaysvortexthawedMgCl2Mg2+concentrationwillbeaffectedbytheamountofDNA,primersandnucleotides,USEMASTERMIXESWHEREPOSSIBLE,,Takenfrom-http://info.med.yale.edu/genetics/ward/tavi/PCR.html,PCROptimization,,FocusontemperatureandMgCl2,THEPERFECTRESULT,APPLICATIONSOFPCR,,基因克隆：基因表達的定量：半定量RT-PCR和實時定量RT-PCT反轉(zhuǎn)錄PCR，RT-PCR快速擴增cDNA末端RACEDNA序列分析,已知序列基因的PCR研究,代表性差異顯示PCR:RDA差異顯示RT-PCR:：DDRT快速擴增多態(tài)性DNA：RAPD,未知序列基因的PCR研究,PCR技術(shù)在基因克隆基因表達差異分析中常用的方法,PCRcloningSemi-quantitativeRT-PCRReal-TimePCR,CloningofPCRproducts,ModifiedprimersbyaddingREsitesatthe5’endofeachprimerT-Astrategy:becausesomethermostaticDNApolymerasecanaddanon-template-dependedAatthe3’endofPCRproducts,socaninsertedthePCRproductsintoaT-vectordiractlyBlunt-endPCRproductscloning,manythermostaticDNApolymerasecanamplifytheDNAcorrectly,InsertedtheDNAproductsintoblunt-REdigestedplasmiddiractly.,,DNA聚合酶的特性及在基因克隆中的應(yīng)用,TaqDNA聚合酶：某些耐熱的DNA聚合酶，在DNA鏈延伸到模板DNA的末端時，并不是立即終止新鏈的延伸，而是在新鏈的3’末端加上一個A，形成突出的3’末端，為基因克隆提供了一個天然的粘末端。,逆轉(zhuǎn)錄酶：來自于逆轉(zhuǎn)錄病毒的逆轉(zhuǎn)錄酶，在以mRNA為模板，反轉(zhuǎn)錄cDNA時，當(dāng)新鏈延伸到模板的末端時，并不是立即終止新鏈的延伸，而是在新鏈的3’末端加上幾個CCCC，形成突出的3’末端，為合成全長的cDNA提供了一個天然接頭位點。,PCRT-Acloning,,,,,,PCR,,,,TT,,,,PCRcloningT-Astrategy,,,,,,,NNNGGATCC,TCTAGAMMM,,,NNNGGATCCYYYCCTAGG,AGATCTMMMTCTAGAWWW,,EcoRI和BglII,,GATCCG,ATCTAG,,,相應(yīng)酶切的克隆質(zhì)粒,PCR克?。和ㄟ^引物對克隆片斷添加酶切位點,,T4DNA酶連接,,,,,,,NNNGGATCC,TCTAGAMMM,,,NNNGGATCCYYYCCTAGG,AGATCTMMMTCTAGAWWW,,EcoRI和BglII,,GATCCG,ATCTAG,,,相應(yīng)酶切的克隆質(zhì)粒,PCR克?。和ㄟ^引物對克隆片斷添加酶切位點,,T4DNA酶連接,RT-PCR,RTase:Avianmyeloblastosisvirus(AMV)andMoloneystrainofmurineleukemiavirus(MMLV).Primersforfirst-strandedcDNAsynthesis:Genespecificprimers,Oligo(dT)primersforbindingthepoly(A)mRNAandRandomhexamerprimers,,RT-PCR,ToamplifycDNAcopiesofRNAToretrieveandclonethe5’and3’terminusofRNATogeneratecDNAlibraryfromaverysmallamountsofmRNAToidentifythemutationsandpolymorphismsTomeasurethestrengthofgeneexpression(semi-quantitativeRT-PCR),,RT-PCR,,FulllengthcDNAsynthesiswithPCR,(SwitchingMechanismAtthe5endoftheRNATranscript)technology,RT-PCR,RTase:Avianmyeloblastosisvirus(AMV)andMoloneystrainofmurineleukemiavirus(MMLV).Primersforfirst-strandedcDNAsynthesis:Genespecificprimers,Oligo(dT)primersforbindingthepoly(A)mRNAandRandomhexamerprimers,,基因表達差異的分析技術(shù),NorthernBlotWesternblotSemi-quantatitiveRT-PCRDNAChipProteinChip二維電泳質(zhì)譜,,NORTHERN,,,,,,,targetgene,internalcontrolgeneactin,GAPDH,RPLP0etc,10X,2X,control,expt,Correctedfoldincrease=10/2=5,Semi-quantitativeRT-PCR,,Correctedfoldincrease=8~10問題:難以準(zhǔn)確設(shè)定PCR擴增的平臺期,Real-TimePCRInstruments,,,,,,dilutionstargetDNA,dilutionsreferenceDNA,,,,,,,C,C,C,C,C,C,E,E,E,E,E,E,,targetprimers,referenceprimers,triplicatescDNA,,triplicatescDNA,,,,Standardcurvemethod,DifferencesbetweenbasicandReal-TimePCRs,Semi-quantitative,notsensitivityPCRproductsvaryfrom100toseveralkbps,Real-timedetection:EachcycleproducesafluorescentsignalAbsolutequantitative,muchmoresensitivityPCRproductsarearound60-150bps,SemiquantatativePCR,Real-TimePCR,,Real-TimePCR---Taqmanprobe,ABIPRISM7000and7700SequenceDetectionSystemReal-timedetection:EachcycleproducesafluorescentsignalproportionaltotheamountofPCRproductpresent,,“Real-Time”PCR,,,,,,F,Q,…...,…...,,,,,,2.Laserexcitesfluorescenttagonprimer.,3.Fluorescenceisabsorbedbyquenchermolecule,4.Detectorregisters“noreaction”.,“Real-Time”PCR,,,,,,F,Q,…...,…...,T,,….,,,,5.PrimerisextendedbyTaqPolymerase,“Real-Time”PCR,,,,,,F,Q,…...,…...,T,,…...,….,,,,6.ExtensioncontinuesuntilthePolymeraseMeetsthefluorescentmolecule.,7.Thepolymerasebreaksdownthetaggedprimer.Thefluorescenttagandquenchermoleculebecomeseparated.,“Real-Time”PCR,,,,,F,Q,…...,T,,…...,,,,…...,…...,…,Q,Q,F,F,8.AsPCRcontinues,thetaggedprimersaredegradedand“free”fluorescentmoleculesaccumulate.,9.Thedetectorrecordsfluorescenceasameasureofamplificationprogress.,Real-TimePCR---SYBRgreen,,,SERIESOF10-FOLDDILUTIONS,IL1-bcon,IL1-bvit,,,,,av=18.03,av=29.63,IL1-beta,Real-TimePCR,TaqmanprobeSpecificprimersFluorescentdyelabeledprobeExpensiveHighspecificity,SRBRgreenSpecificprimersNoprobesCheaperReliable,,HistoryofPCR,PCR技術(shù)是由美國科學(xué)家KaryMullis于1983年發(fā)明的。由于當(dāng)時沒有耐熱DNA聚合酶，因此PCR過程中每個循環(huán)都需要添加DNA聚合酶，PCR技術(shù)操作十分煩瑣而且不實用。PCR技術(shù)的自動化歸因另外2個重大發(fā)現(xiàn)：耐熱DNA聚合酶的發(fā)現(xiàn)，和計算機控制的熱循環(huán)儀（DNAthermalcycler)的出現(xiàn)。TaqDNA聚合酶被美國《Science》雜志評為1993年的明星分子。,HistoryofPCR,1987年P(guān)CR技術(shù)得到美國專利局的專利授權(quán)。1989年DuPont公司對該專利提出異議：PCR技術(shù)應(yīng)當(dāng)是公共產(chǎn)權(quán)。斯坦福大學(xué)的KornbergHG也對PCR技術(shù)專利提出異議。理由是認(rèn)可具有生物學(xué)知識的人都可以從Kornberg的文章推知如何操作PCR。1993年MullisKary獲得諾貝爾獎。,HistoryofPCR,MilestonesofPCR,,DNApolymerase:KornbergA.(1957)Oligonucleotidesynthesis:KhoranaHG(1967-1968)ThePCRidea:KhoranaHG(1971,J.Mol.Biol)Theidea:KaryMullis(1983)ThermostableDNApolymerase(1989)AutomatedThermalcyclerInnovativeapplicationsReal-TimePCRRACEFlowChipPCR,HistoryofPCR,LouisPasteur:Onceremarkedthat"chancefavorsthepreparedmind,"andcertainlythehistoryofscientificprogresssupportshiscontention,suchasNewtonsdiscoveryofgravityfollowinghisencounterwithanapple,Flemingsdiscoveryofpenicillinonacontaminatedpetridish.KaryMullis:Scientiststodaycontinuetotakeunexpectedturnsontheirpathstodiscovery.Onesuchrecentdetouroccurredin1983onU.S.Route101innorthernCalifornia.,HistoryofPCR,KaryMulliswasbornin1944inNorthCarolina.HeobtainedhisBachelorsdegreeinChemistryin1966fromtheGeorgiaInstituteofTechnologyandin1972receivedaPhDinBiochemistryfromtheUniversityofCaliforniaatBerkeley.HewasthenofferedatechnicianspostattheCetusCorporationofEmeryvillein1978.In1983,whilstdrivingalongthehighwayfromSanFranciscotohishomeinLaJolla,California,MulliswasthinkingaboutasimplemethodofexponentiallyamplifyingaDNAsequenceinatesttube.MullisthentookhisconcepttohisassociatesatCetusandtogethertheytooktheideaandmadeitworkinanexperimentalsystem,Mulliswasawarded10,000US$forhisidea.DuetotheunprecedentedpopularityofthetechniqueanditsrevolutionaryimpactonMolecularBiology,KaryMulliswasawardedtheNobelPrizeforChemistryin1993.,HistoryofPCR,,TheDNAcomplexwouldbedenaturedtoformsinglestrands,thisdenaturationstepwouldbecarriedoutinthepresenceofasufficientlyLargeexcessofthetwoappropriateprimers……..DNApolymerasewillbeaddedtocompletetheprocessofrepair…..thewholecyclewouldberepeated(H.G.Khorana,1971;JournalofMolecularBiology,56:341IstoppedthecaragainandstarteddrawinglinesoftheDNAmoleculeshybridizingandextending,theproductofonecyclebecomingthetemplatesforthenextinachainreaction….(K.B.Mullis,1990,ScientificAmerican,262.56,Continuous-FlowPCRonaChip,Science,280(5366),1046-1048,15May1998,PolymeraseChainReactionPCRProteinChainReactionPCR,WhowillbethenextKaryMullis?,KhoranaHG.1971;JournalofMolecularBiology,56:341:DNAPCRMullisK.1983;PCR,TellingGC.2001;Protein-basedPCR,NatureMedicine,2001Jul;7(7):778-9Who?ProteinPCR,