(自学)体外定点突变

体外定点突变Why?WanttodeterminehowDNAand/orencodedproteinsfunctioninintactentity(virus,bacterium,cell,animaletc.)MostdirectwaytofindoutwhatageneorproteindoesistofindoutwhathappenswhenitismissingormutatedStudymutantsthatlackgene/proteinorexpressalteredversionofit-determinewhichbiologicalprocessesarealteredinmutantsChangesolubility,stability,structure,functionofaproteinChangeenzymeactivityorsubstratespecificityHow?GENETICSCREEN…SelectPHENOTYPE–determineGENOTYPEorREVERSEGENETICS…CreateGENOTYPE–determinePHENOTYPEi.e.,translatesequenceintofunctionTypesofMutation(1)POINTMUTATION:mapstosinglesiteingenome,correspondingtosinglenucleotidepairorverysmallpartofsinglegeneINVERSIONMUTATION:invertssegmentofchromosomeDELETION:deletessegmentofchromosomeTRANSLOCATION:breaksoffsegmentfromonechromosomeandattachesittoanotherTypesofMutation(2)LETHALMUTATION:causesdevelopingorganismtodieprematurelyCONDITIONALMUTATION:producesitsphenotypiceffectonlyundercertainconditions,calledtherestrictiveconditions.Underotherconditions—thepermissiveconditions—theeffectisnotseen.Foratemperature-sensitivemutation,therestrictiveconditiontypicallyishightemperature,whilethepermissiveconditionislowtemperatureLOSS-OF-FUNCTIONMUTATION:eitherreducesorabolishestheactivityofthegene.Thisisthemostcommonclassofmutation.Loss-of-functionmutationsareusuallyrecessive—theorganismcanusuallyfunctionnormallyaslongasitretainsatleastonenormalcopyoftheaffectedgeneTypesofMutation(3)NULLMUTATION:loss-of-functionmutationcompletelyabolishingactivityofgeneGAIN-OF-FUNCTIONMUTATION:increasesactivityofgeneormakesitactiveininappropriatecircumstances;usuallydominantDOMINANTNEGATIVEMUTATION:dominant-actingmutationblockinggeneactivity,causesloss-of-functionphenotypeeveninpresenceofnormalcopyofgene.OccurswhenmutantgeneproductinterfereswithfunctionofnormalgeneproductSUPPRESSORMUTATION:suppressesphenotypiceffectofanothermutation,sodoublemutantseemsnormal:InVitroMutagenesis(体外基因突变)Atitsmostsimplistic,invitromutagenesisallowsustochangethebasesequenceofaDNAsegmentorgeneMutationscanbelocalizedorgeneral,randomortargeted;LessspecificmethodsofmutagenesisusedtoanalyzeregulatoryregionsofgenesMorespecificmethodsofmutagenesisusedtounderstandcontributionofindividualaminoacids,orgroupsofaminoacids,tostructureandfunctionoftargetproteinBothmethodsgeneratemutantsinvitro,withoutphenotypicselection体外基因突变(InVitroMutagenesis)包括：单个碱基或片断的替换基因片断的插入基因片断的删除根据其特点可将体外基因突变技术分为两大类：位点特异性突变定点突变随机突变位点特异性突变的类型寡核苷酸介导的基因突变指用含有突变碱基的寡聚核苷酸片断作为引物，在聚合酶的作用下启动DNA分子进行复制。盒式突变是利用一段人工合成的含基因突变序列的寡核苷酸片段，取代野生型基因中的相应序列。PCR介导的基因突变DesignofMutagenicOligonucleotidesCrucialstepinsite-directedmutagenesisisdesignofmutagenicoligoBydefinition,oligosequenceincorporatesatleastonebasechangeMaybefarmorecomplicatedincludinginsertions,deletionsandcompoundsubstitutionsMinimumlengthofoligodeterminedbycomplexityofmutationSimplesinglebasemutationsuse~25ntoligosMorecomplicatedmutationsmayrequireoligosof80ntsEfficiencyofmutagenesisoptimizedbydesignof;BasesequenceBasecompositionMeltingtemperaturePropensitytoformsecondarystructuresSpecificityofannealingClassicalSite-DirectedMutagenesisProtocolsforsite-directedmutagenesisinvolve;DesignandsynthesisofmutagenicoligonucleotidesHybridizationofmutagenicoligotossDNAtargetclonedintobacteriophagevector(usuallyM13)–eliminatescompetitionbetweenmutagenicoligoandcomplementaryDNAstrandExtensionofhybridizedoligobyDNApolymerasewithall4dNTPsFormationofclosedcircularDNAbyligationwithDNAligaseTransfectionofsusceptiblebacteriaScreeningformutantclonesbyhybridization(e.g.,usingoligo)–sequenceconfirmationRecoveryofmutatedDNAfragmentSubstitutionofmutagenizedfragmentforcorrespondingwtDNAsequenceTraditionalgeneralprocedureGeneratessDNA(M13)AnnealmutagenicoligoExtendwithDNApolymeraseanddNTPSealnickswithDNAligaseSelectformutantstrandordeselectforwildtypestrandTransformandscreenIsolateDNAandsequencetoverify1.Cloneinsertintoplasmidvector2.Denatureandannealmutagenicoligonucleotides3.ExtendusingDNApolymerase;LigateusingT4DNAligase4.Selectmutantstrand;RetransformintofinalhostClassicalSite-DirectedMutagenesisProportionofclonescontainingmutation0.1%-50%dependingonefficiencyofmutagenicoligoLowefficiencySeveralmethodsdevelopedtoselectagainstclonescontainingnon-mutantDNAincluding;SelectivedigestionoftemplateDNAsbyremovalofmodifiedbases(KUNKELMUTAGENESIS),EliminationofrestrictionsitefrommutantDNAs(USEMUTAGENESIS)SelectivedigestionoftemplateDNAsbyprotectionofmodifiedbases(Phosphorothiatemethod),usingrestrictionenzymesorexonucleasesKunkel法原理:在E.coli中dUTPdUMP在dUTP酶缺失体中（dut-）dUTPdUMP在正常情况下，尿嘧啶－Ｎ－糖基化酶(ung-)可以去除掺入DNA中的尿嘧啶残基。但在ung-的菌株中，此酶失活。因此在大肠杆菌dut-ung-菌株中生长的M13噬菌体的DNA中将含有20-30个尿嘧啶残基。用这些噬菌体感染ung+菌株，尿嘧啶被迅速去除，DNA链遭到破坏，感染力下降约5个数量级KunkelMutagenesisTakesadvantageofstrongselectionagainsturacil-substitutedDNAbyE.colistrainsexpressinguracil-DNAglycosylaseSingle-strandedtemplateDNApreparedbygrowthofrecombinantbacteriophageM13indut-(lackingdUTPase)ung-(lackinguracilglycosylase)F’E.colistrainStandar