研究生課程：PCR技術(shù)的新進(jìn)展及其應(yīng)用.ppt

KaryMullis1983,POLYMERASECHAINREACTION,AlicencetodomolecularbiologyAkeycentraltechniquethathasrevolutionisedmolecularandconsequentlycellbiology,InthisLecture,PrincipleofPCRPCR,thebasicsOptimizethePCRandtroubleshootApplicationofPCRReverse-TranscriptionPCR（反轉(zhuǎn)錄PCRPCRcloning（PCR克隆技術(shù)Real-TimePCR（實(shí)時(shí)定量PCRHistoryofPCR,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(duì)復(fù)雜生命過程的一種簡(jiǎn)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,25or50lsinamicroEppendorf(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),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Startingnucleicacid-DNA/RNATissue,cells,blood,hairroot,saliva,semenThermo-stableDNApolymerasee.g.TaqpolymeraseOligonucleotidesDesignthemwell!BufferTris-HCl(pH7.6-8.0)Mg2+dNTPs(dATP,dCTP,dGTP,dTTP),OPTIMISINGPCRTHEREACTIONCOMPONENTS,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),OPTIMISINGPCRTHEREACTIONCOMPONENTS,Length18-30nt(21nt)Basecomposition;50-60%GCrichpairsshouldhaveequivalentTmsTm=(numberofA+Tresidues)x2C+(numberofG+Cresidues)x4CInitialuseTm5CAvoidinternalhairpinstructuresnosecondarystructureAvoidaTatthe3endAvoidoverlapping3endswillformprimerdimersCanmodify5endstoaddrestrictionsitesetc,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),OPTIMISINGPCRTHEREACTIONCOMPONENTS,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(dá)的定量：半定量RT-PCR和實(shí)時(shí)定量RT-PCT反轉(zhuǎn)錄PCR，RT-PCR快速擴(kuò)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(kuò)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(dá)差異分析中常用的方法,PCRcloningSemi-quantitativeRT-PCRReal-TimePCR,CloningofPCRproducts,ModifiedprimersbyaddingREsitesatthe5endofeachprimerT-Astrategy:becausesomethermostaticDNApolymerasecanaddanon-template-dependedAatthe3endofPCRproducts,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(shí)，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(gè)A，形成突出的3末端，為基因克隆提供了一個(gè)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(shí)，當(dāng)新鏈延伸到模板的末端時(shí)，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(gè)CCCC，形成突出的3末端，為合成全長(zhǎng)的cDNA提供了一個(gè)天然接頭位點(diǎn)。,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(duì)克隆片斷添加酶切位點(diǎn),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克隆：通過引物對(duì)克隆片斷添加酶切位點(diǎn),T4DNA酶連接,RT-PCR,RTase:Avianmyeloblastosisvirus(AMV)andMoloneystrainofmurineleukemiavirus(MMLV).Primersforfirst-strandedcDNAsynthesis:Genespecificprimers,Oligo(dT)primersforbindingthepoly(A)mRNAandRandomhexamerprimers,RT-PCR,ToamplifycDNAcopiesofRNAToretrieveandclonethe5and3terminusofRNATogeneratecDNAlibraryfromaverysmallamountsofmRNAToidentifythemutationsandpolymorphismsTomeasurethestrengthofgeneexpression(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(dá)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=810問題:難以準(zhǔn)確設(shè)定PCR擴(kuò)增的平臺(tái)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(guó)科學(xué)家KaryMullis于1983年發(fā)明的。由于當(dāng)時(shí)沒有耐熱DNA聚合酶，因此PCR過程中每個(gè)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(shí)用。PCR技術(shù)的自動(dòng)化歸因另外2個(gè)重大發(fā)現(xiàn)：耐熱DNA聚合酶的發(fā)現(xiàn)，和計(jì)算機(jī)控制的熱循環(huán)儀（DNAthermalcycler)的出現(xiàn)。TaqDNA聚合酶被美國(guó)Science雜志評(píng)為1993年的明星分子。,HistoryofPCR,1987年P(guān)CR技術(shù)得到美國(guó)專利局的專利授權(quán)。1989年DuPont公司對(duì)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(duì)PCR技術(shù)專利提出異議。理由是認(rèn)可具有生物學(xué)知識(shí)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(jiǎng)。,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,