PCR技术及实用方法

()METHODSINLABOFMOLECULARPLANTBREEDING(2)PCR1,2　11,,5720252,,350019PCR,、PCR。PCR。,。PCR,PCRTechniqueanditsPracticalMethodsZhengJingsheng1,2　LǜBei11HainanProvincialKeyLaboratoryofCropMolecularBreeding,Sanya,5720252TheInstituteofRice&wheat,FujianAcademyofAgricalturalSciences,Fuzhou,350019ABSTRACTThispaperintroducedPCRprinciples,reactioncomponents,reactionprogramsandelectrophoresisdetectingtechniques.AllofthepracticalprotocolswerederivedfrommanyfamousdomesticandinternationallaboratoriesandmodifiedbyHainanProvincialKeyLaboratoryofCropMolecularBreeding(HiCMBL).Ithasmorebeneficialtoadaptthoseprotocolsastechnologyplatforminthelabofmolecularplantbreedingduetoitsgoodpracticability,whichcouldmeetthedemandsoftheresearchesinfieldsofmolecularbiologyandbiotechnology.KEYWORDSPCR,Practicalmethod,Molecularplantbreeding1PCR,(PolymeraseChainReaction,PCR)1985PE-CetusKaryBanksMulisDNA。DNA,DNA,DNA。PCR、,、、,,2003,1,3,381—394MolecularPlantBreeding,2003,Vol.1,No.3,381—394　。KaryBanksMulis1994。PCR:DNADNA,DNA,DNA,。,DNA3'-OH,,5'※3',DNA。PCR:DNA(DNA)、、4(dNTPs)、DNA。PCR,DNA(denaturation)、(anneal-ing)(extension)。,。PCRDNA,。2PCR2.1DNAPCRDNA,DNAcDNA,mRNAPCR,mRNAcD-NA。PCR,DNA,、、DNA、DNA。DNA,,PCRSDSTaqDNA,PCR。,DNA10mmol/LTris-HCl(pH7.6)、0.1mmol/LEDTA(pH8.0)。PCRDNA(),DNA30-50ng,1DNAPCR,1DNADNA。PCR40(HPV)DNA,,DNAPCR。2.2PCRDNA,10-30。,5'3',DNAPCR。,171。,16,PCR。,PCR。PCR。DNA,DNA。,、。G+C(G+C%),GC,GC。,,G+C40%-60%,(meltingtemperature,Tm)55℃。,。,3',,3'2,“”。DNA。,DNA3',3'5-6DNA、。,。(,2002)。4,,,,。“”。,PCRPCGENE、DNAMan,DNA382　　　MolecularPlantBreeding。,25%4℃-20℃,1-2。PCR,0.1-1(mol/L。,,,30,PCR。DNADNA(HighcomplexityDNA)DNA,;DNADNA(LowcomplexityDNA)DNA,。RAPD、ISSRSSR,PromegaInvitrogen。2.3dNTPsdNTPs(dATP、dCTP、dGTPdTTP)PCRDNA。PCR50-200μmol/L,10-15μmol/L。PCR,dNTP50mmol/LTaq;。4dNTP,,,。100μl,50μmol/L200μmol/LdNTP6.5μg25μgDNA。dNTPsPromega。25mmol/ldNTPs,4。2.4DNADNADNAIKlenow。3'※5',,。Klenow,DNA,PCR,PCR、、。,—(Thermusaquatics,Taq)60-68kDa,2,000-8,000U/mgDNA。94kDa,200,000U/mgDNA,2496,832。95℃,PCR,PCR,PCR,PCR、,PCR。,TaqDNAPCR。70-75℃,72℃TaqDNA(94kDa)DNA,150。,TaqDNA,。92.5℃、95℃97.5℃,PCRTaqDNA130min、40min5-6min50%。92-94℃,95℃。100μlPCR,TaqDNA1-2.5U,DNA,TaqDNA1-4U。PCR,PCR。TaqDNAMg2+,Mg2+。TaqDNA3'※5',,2×10-4。,PCR。PCR,DNA()。,。,DNAPCR,PfuDNA。PfuDNADNA,Pyrococcusfuriosis,90kDa,PCR、、、PCRPCRTechniqueanditsPracticalMethods　383　　。Pfu5'※3'DNA,3'※5',5'※3'(),PCR,600bp/min,65-75℃,dNTP100-300μmol/l,Mg2+2-3mmol/l,pH8.1-9.1,5U。Taq,PfuTaq,Pfu3'※5'(),Pfu。Pfu,,18bp,Tm55-58℃,0.1-0.5μmol/l,Taq。Pfu3'※5'(dNTP),Pfu,PCR。PfuTaq。GC98℃,Pfu。PfuPCR,T/A,PCR3'-AT/A。100μl5UPfu。,PfuTaqDNA。TaqDNA、PfuDNA,DNA:TthDNA:5'※3'5'※3',、PCRRT-PCR;TflDNA:5'※3'5'※3',,PCR、DNA;TliDNA:5'※3'3'※5',PCR;PlatiumPfxDNA:5'※3'3'※5',TaqDNA,PCR、PCRPCR;PlatiumDNA():5'※3'3'※5',TaqDNA,PCR、PCRPCR。2.5PCR10mmol/LTris-HCl(pH8.3),50mmol/LKCl1.5mmol/LMgCl2。72℃,pH1,pH7.2(,1999),、,,(Krawetzetal.,1989)。10×PCRMg2+Mg2+,Mg2+,25mmol/lMgCl2,。Mg2+10×PCR0.1mol/LTris-HCl(pH8.0),0.5mol/lKCl1%TritonRX-100。2.6Mg2+PCRMg2+,Mg2+dNTP0.5-2.5mmol/L。dNTP0.2mmol/L,Mg2+1mmol/L。Mg2+,;,PCR。Mg2+EDTA、,Mg2+Mg2+。,、、dNTP(),Mg2+。,KCL(50mmol/L)Tris-HCl(10mmol/L),MgCl2(0.05-5mmol/L,0.5mmol/L),,,Mg2+(,1999)。Mg2+(MgCl2·6H2O),Sigma,MgCl2,,。20mmol/lMgCl2,4.066g,800mlddH2O,1L,。KlenowPCR10%(DMSO),DNA,GC。TaqDNAPCR,DMSO,DMSOTaqDNA,。,DMSOPCR。PCR,,,PCR384　　　MolecularPlantBreeding。3PCRPCR()PCR、。PCR(denaturation)、(an-nealing)(extension)。,PCRPCR。3.1DNA。、,,。,DNA,PCR;,、,。95℃,30,GCDNA。(Tm),DNA。TmDNAG+C,Tm=81.5-16.6lg[Na+]+0.41(G+C)%-600/N,N(,1999)。DNAPCR,PCR。3.2DNA。,PCR。、。(Tm)5℃,37-55℃,1min。,PCR(,2002)。Tm:20,Tm=2℃×(A+T)+4℃×(G+C);20,Tm=81.5+0.41(G+C)%-600/L,L。GC50%、2055℃。3.3DNA3-OH,。DNA。TaqDNA,70-75℃。72℃,1min2kb。、,1-3min。、、,;,。,,。,PCR(1nmol/L),,,。1kb,1min。100-300,、。TaqDNA,。,PCR,。3.4DNA、25-40。PCR,DNA。1。,,;,PCR。1　Table1OptimumcyclingnumberofdifferenttargetDNAmoleculesNo.oftargetDNAmoleculeCyclingnumber3×10525-305×10430-351×10335-405040-45PCRPCRTechniqueanditsPracticalMethods　385　　4PCRPCR10-100μl,。ddH2O、PCR、Mg2+、dNTPs、TaqDNA,、,,Eppendorf,DNA。Eppendorf,。PCREppendorf,PCRPCR。PCR,,。PCR,dNTPs、TaqDNAPromega,、Mg2+、Buffer;RAPD、ISSRBritishColumbia,、SSRIn-vitrogen,、。4.1RAPD4.1.1RAPD(randomamplifiedpolymorphismicDNA,RAPD),9-10,PCR。DNA。,PCR,DNA。RAPD,(BritishColumbia)。RAPD,RFLP。RAPD:DNA,DNA;,;,。:,,,(,2001)。4.1.2:ReactionComponentsStockSolutionConcentrationFinalConcentration(μl)WorkingSolutionPreparationddH2O——17.6PCRbuffer10×PCRBuffer1×PCRBuffer2.5Mg2+15mmol/l1.5mmol/l1.5dNTPs25mmol/l0.2mmol/l0.23.75μmol/l0.15μmol/l1.0Taq5U/μl1U/μl0.2DNA7.5-12.5ng15-25ng2.0——25.0:Step　1:94℃5minStep　2:94℃45sStep　3:36℃37℃60sStep　4:72℃2minstep　52-4,45step　6:72℃7minstep　7:4℃1-12hstep　84.2ISSR4.2.1ISSR(inter-simplesequencerepeats,ISSR)DNA。ISSRSSRDNA。SSR,SSRPCR,、SSRDNA。3'5'2-4,,SSR,ISSR。ISSRPCR20,PCR,RAPD。ISSR,(,2001)。386　　　MolecularPlantBreeding4.2.2:ReactionComponentsStockSolutionConcentrationFinalConcentration(μl)WorkingSolutionPreparationddH2O——17.6PCRbuffer10×PCRBuffer1×PCRBuffer2.5Mg2+15mmol/l1.5mmol/l1.5dNTPs25mmol/l0.2mmol/l0.25μmol/l0.2μmol/l1.0Taq5U/μl1U/μl0.2DNA15ng30ng2.0——25.0:step　1:94℃5minstep　2:94℃30sstep　3:37-67℃1minstep　4:72℃2minste