高效快速的点突变方法.ppt

高效快速的点突变方法,陈严姜明汤敏谦,宝生物工程（大连）有限公司,116600,目的：1.在目的基因中的特定位点导入突变，包括：,碱基缺失碱基置换碱基插入,2.通过突变基因的表达，可以为蛋白质结构及功能的分析和改造提供有效途径。,TaKaRa用于定点突变的试剂盒,Mutan-ExpressKmKitMutan-SuperExpressKmKitMutan-KKitTaKaRaMutanBESTKit,Mutan-ExpressKm,原理：ODA法,E.coliBMH71-18mutS、MV1184CompetentCellorElectroCell导入变异Primer,材料与方法,Materials,Protocol,ReactionMixture,Annealing/Extension,DNASample,Transformation（1）E.coliBMH71-18mutS,PlasmidMiniprep,DNA,Transformation（2）MV1184,Colony,Sequencing,Mutan-SuperExpressKm,原理：ODA-LAPCR法,E.coliMV1184CompetentCellorElectroCellpKF18k/pKF19kDNA导入变异用寡聚核苷酸（5-P）,材料与方法,Materials,ReactionMixture,PCR反应,DNA精制,Transformationsup0菌株（MV1184）,Sequencing,Colony,Protocol,Mutan-K,原理：Kunkel法,材料与方法,E.coliBMH71-18mutS、CJ236、MV1184CompetentCellorElectroCell导入变异用寡聚核苷酸M13mp18RForM13mp19RForpUC118/119,Materials,Protocol,ClonedDNA（M13）,TransfectionCJ236,ssDNASample,变异用寡聚核苷酸,5磷酸化,Annealing,Extension,DNASample,TransfectionBMH71-18mutS,Phage,TransfectionMV1184（ung+Cell）,噬菌斑,TaKaRaMutanBEST,原理：PCR法,材料与方法,导入变异用PrimerE.coliJM109Competentcell,Materials,Protocol,ReactionMixture,PCR反应,电泳回收PCRFragment,BKL反应,DNASample,Transformation（JM109）,Sequencing,Colony,TaKaRaMutanBESTKit使用实例,5GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTG33CTTAAGCTCGAGCCATGGGCCCCTAGGAGATCTCAGCTGGAC5,pUC118,引入点变异区域碱基序列,GGATCCTCTAGAGTCGACCTG,CTTAAGCTCGAGCCATGGGCC,Primer1,Primer2,StepI.PCR反应,10PyrobestBufferdNTPMixtrue（各2.5mM）Primer1Primer2TemplatePyrobestDNAPolymerase(5U/ml)灭菌蒸馏水upto,5l4l0.21.0M(finalconc.)0.21.0M(finalconc.)1g0.25l50l,9430sec5530sec725min,30Cycles,PCRFragment10BluntingKinationBufferBluntingKinationEnzymeMix.ddH2Oupto,2l1l20l,0.2-20pmol,Step.BluntingKinationReaction,37，10min.,Step.LigationReaction,DNAFragment5lLigationSolutionI5l,16,1hrTransformationJM109Colony,Sequencing,结果：,1.Transformation,2.Sequencing挑