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首页 DNA sequencing with chain-terminating inhibitor…

DNA sequencing with chain-terminating inhibitors.pdf

DNA sequencing with chain-termi…

上传者: susuxuxu 2011-04-05 评分 0 0 0 0 0 0 暂无简介 简介 举报

简介:本文档为《DNA sequencing with chain-terminating inhibitorspdf》,可适用于人文社科领域,主题内容包含ProcNatiAcadSciUSAVol,No,pp,DecemberBiochemistryDNAsequencingwithchainterm符等。

ProcNatiAcadSciUSAVol,No,pp,DecemberBiochemistryDNAsequencingwithchainterminatinginhibitors(DNApolymerasenucleotidesequencesbacteriophageX)FSANGER,SNICKLEN,ANDARCOULSONMedicalResearchCouncilLaboratoryofMolecularBiology,CambridgeCBQH,EnglandContributedbyFSanger,October,ABSTRACTAnewmethodfordeterminingnucleotidesequencesinDNAisdescribedItissimilartothe"plusandminus"methodSanger,FCoulson,AR()JMolBiol,butmakesuseofthe','dideoxyandarabinonucleosideanaloguesofthenormaldeoxynucleosidetriphosphates,whichactasspecificchainterminatinginhibitorsofDNApolymeraseThetechniquehasbeenappliedtotheDNAofbacteriophagebXandismorerapidandmoreaccuratethaneithertheplusortheminusmethodThe"plusandminus"method()isarelativelyrapidandsimpletechniquethathasmadepossiblethedeterminationofthesequenceofthegenomeofbacteriophageX()ItdependsontheuseofDNApolymerasetotranscribespecificregionsoftheDNAundercontrolledconditionsAlthoughthemethodisconsiderablymorerapidandsimplethanotheravailabletechniques,neitherthe"plus"northe"minus"methodiscompletelyaccurate,andinordertoestablishasequencebothmustbeusedtogether,andsometimesconfirmatorydataarenecessaryWMBarnes(JMolBiol,inpress)hasrecentlydevelopedathirdmethod,involvingribosubstitution,whichhascertainadvantagesovertheplusandminusmethod,butthishasnotyetbeenextensivelyexploitedAnotherrapidandsimplemethodthatdependsonspecificchemicaldegradationoftheDNAhasrecentlybeendescribedbyMaxamandGilbert(),andthishasalsobeenusedextensivelyforDNAsequencingIthastheadvantageovertheplusandminusmethodthatitcanbeappliedtodoublestrandedDNA,butitrequiresastrandseparationorequivalentfractionationofeachrestrictionenzymefragmentstudied,whichmakesitsomewhatmorelaboriousThispaperdescribesafurthermethodusingDNApolymerase,whichmakesuseofinhibitorsthatterminatethenewlysynthesizedchainsatspecificresiduesPrincipleoftheMethodAtkinsonetal()showedthattheinhibitoryactivityof','dideoxythymidinetriphosphate(ddTTP)onDNApolymeraseIdependsonitsbeingincorporatedintothegrowingoligonucleotidechainintheplaceofthymidylicacid(dT)BecausetheddTcontainsno'hydroxylgroup,thechaincannotbeextendedfurther,sothatterminationoccursspecificallyatpositionswheredTshouldbeincorporatedIfaprimerandtemplateareincubatedwithDNApolymeraseinthepresenceofamixtureofddTTPanddTTP,aswellastheotherthreedeoxyribonucleosidetriphosphates(oneofwhichislabeledwithp),amixtureoffragmentsallhavingthesame'andwithddTresiduesatthe'endsisobtainedWhenthismixtureisfractionatedbyelectrophoresisondenaturingacrylamidegelsthepatternofbandsshowsthedistributionofdTsinthenewlysynthesizedDNAByusinganalogousterminatorsfortheothernucleotidesinseparateincubationsandrunningthesamplesinparallelonthegel,apatternofbandsisobtainedfromwhichthesequencecanbereadoffasintheotherrapidtechniquesmentionedaboveTwotypesofterminatingtriphosphateshavebeenusedthedideoxyderivativesandthearabinonucleosidesArabinoseisastereoisomerofriboseinwhichthe'hydroxylgroupisorientedintranspositionwithrespecttothe'hydroxylgroupThearabinosyl(ara)nucleotidesactaschainterminatinginhibitorsofEscherichiacoliDNApolymeraseIinamannercomparabletoddT(),althoughsynthesizedchainsendingin'araCcanbefurtherextendedbysomemammalianDNApolymerases()InordertoobtainasuitablepatternofbandsfromwhichanextensivesequencecanbereaditisnecessarytohavearatioofterminatingtriphosphatetonormaltriphosphatesuchthatonlypartialincorporationoftheterminatoroccursForthedideoxyderivativesthisratioisabout,andforthearabinosylderivativesaboutMETHODSPreparationoftheTriphosphateAnaloguesThepreparationofddTTPhasbeendescribed(,),andthematerialisnowcommerciallyavailableddAhasbeenpreparedbyMcCarthyetal()WeessentiallyfollowedtheirprocedureandusedthemethodsofTener()andofHoardandOtt()toconvertittothetriphosphate,whichwasthenpurifiedonDEAESephadex,usingaMgradientoftriethylaminecarbonateatpHThepreparationofddGTPandddCTPhasnotbeendescribedpreviouslyhoweverweappliedthesamemethodasthatusedforddATPandobtainedsolutionshavingtherequisiteterminatingactivitiesTheyieldswereverylowandthiscanhardlyberegardedasadequatechemicalcharacterizationHowever,therecanbelittledoubtthattheactivitywasduetothedideoxyderivativesThestartingmaterialfortheddGTPwasNisobutyryl'OmonomethoxytrityldeoxyguanosinepreparedbyFEBaralle()Aftertosylationofthe'OHgroup()thecompoundwasconvertedtothe','didehydroderivativewithsodiummethoxide()TheisobutyrylgroupwaspartlyremovedduringthistreatmentandremovalwascompletedbyincubationinNH(specificgravity)overnightatThedidehydroderivativewasreducedtothedideoxyderivative()andconvertedtothetriphosphateasfortheddATPThemonophosphatewaspurifiedbyfractionationonaDEAESephadexcolumnusingatriethylaminecarbonategradient(M)butthetriphosphatewasnotpurifiedddCTPwaspreparedfromNanisoyl'Omonomethoxytrityldeoxycytidine(CollaborativeResearchInc,Waltham,MA)bytheabovemethodbutthefinalpurificationonDEAESephadexwasomittedbecausetheyieldwasverylowandthesolutioncontainedtherequiredactivityThesolutionwasuseddirectlyintheexperimentsdescribedinthispaperAnattemptwasmadetopreparethetriphosphateoftheintermediatedidehydrodideoxycytidinebecauseAtkinsonetAbbreviations:ThesymbolsC,T,A,andGareusedforthedeoxyribonucleotidesinDNAsequencestheprefixddisusedforthe','dideoxyderivatives(eg,ddATPis','dideoxyadenosine'triphosphate)theprefixaraisusedforthearabinoseanaloguesProcNatlAcadSciUSA()A()Ad(GATCGATC:S:<||TIAxaAC,triIAGAC~~t'~(AG("QiTteOzcCTaGA:eAA(AT(,Q(AA)((oTIAACTTAATAAQ(oAGrTTA*OFOmPtA'AAAIT,ITATAAAUAs)jij~~~C'GAATcAtA($(tittojATbyCCAox~~~~~~~(ACAAAdGATACsiAA~ACTGACAAAAAACCCAAFIGAutoradiographoftheacrylamidegelfromthesequencedeterminationusingrestrictionfragmentsAdandAasprimersonthecomplementarystrandofIXDNATheinhibitorsusedwere(lefttoright)ddGTP,ddATP,ddTTP,andaraCTPElectrophoresiswasonaacrylamidegelatmAforhrThetopcmofthegelisnotshownTheDNAsequenceiswrittenfromlefttorightandupwardsbesidethecorrespondingbandsontheradioautographThenumberingisasgiveninrefal()haveshownthatthedidehydrodideoxyTTPisalsoactiveasaterminatorHowever,wewereunsuccessfulinthisThesecompoundsseemmuchlessstablethanthedideoxyderivativesaraATPandaraCTPwereobtainedfromPLBiochemicalsInc,Milwaukee,WISequencingProcedureRestrictionenzymefragmentswereobtainedfromOXreplicativeformandseparatedbyelectrophoresisonacrylamidegelsThematerialobtainedfrom,ugofOXreplicativeformin,AlofHwasmixedwithAlofviralorcomplementarystrandXDNA(jg)andMiofHXbuffer()andsealedinacapillarytube,heatedtoformin,andthenincubatedatforminThesolutionwasdilutedtoAlwithHbufferandAlsamplesweretakenforeachincubationandmixedwithMloftheappropriate"mix"andilofDNApolymerase(accordingtoKlenow,Boehringer,Mannheim)(units)EachmixcontainedXHbuffer,MCiofaPdATP(specificactivityapproximatelymCi,Mnwol)andthefollowingothertriphosphatesddT:mMdGTP,mMdCTP,mMdTTP,mMddTTPddA:mMdGTP,mMdCTP,mMdTTP,mMddATPddG:mMdCTP,mMdTTP,mMdGTP,mMddGTPddC:mMdGTP,mMdTTP,mMdCTP,approximatelymMddCTP(TheconcentrationoftheddCTPwasuncertainbecausetherewasinsufficientyieldtodetermineit,buttherequireddilutionofthesolutionwasdeterminedexperimentally)araC:mMdGTP,mMdTTP,mMdCTP,mMaraCTPIncubationwasatroomtemperatureforminThenMlofmMdATPwasaddedandincubationwascontinuedforafurtherminIfthisstep(chase)wasomittedsometerminationatAresiduesoccurredinallsamplesduetothelowconcentrationoftheaPldATPWithsmallprimers,whereitwasunnecessarytocarryoutasubsequentsplitting(asintheexperimentshowninFig),thevariousreactionmixturesweredenatureddirectlyandappliedtotheacrylamidegelforelectrophoresis()Iffurthersplittingwasnecessary(seeFig),MiloftheappropriaterestrictionenzymewasaddedshortlyafterthedATP"chase,andincubationwasatThesinglesiteribosubstitutionprocedure(NLBrown,unpublished)wascarriedoutasfollowsTheannealingoftemplateandprimerwascarriedoutasabovebutin"Mnbuffer"(mMTrisCl,pHmMmercaptoethanolBiochemistry:'SangeretalProcNatiAcadSciUSA()GATCTaCddCGA~I~ATiTTCSiECGTTCAohsPAGACAGAACAACGAGACCATATGTTT=*GGACGAAAA~~AACTG^cGAAG)*CCCCCBlitzAACC*ATAGI~~~CAsC=M~AAWT'A*C~~~~~AATGTT'^cAcAAACGTTATFIGAutoradiographfromanexperimentusingfragmentRasprimeronthecomplementarystrandofpXDNAConditionswereasinFigwiththefollowingexceptions:ddCTPwasusedasinhibitorinsteadofaraCTPAfterincubationofthesolutionsatroomtemperatureformin,glofmMdATPandglofrestrictionenzymeHaeIII(units)wereaddedandthesolutionswereincubatedatforminTheHaeIIIcutsclosetotheHindIIsiteanditwasusedbecauseitwasmorereadilyavailableTheelectrophoresiswasonaacrylamidegelatmAforhrThetopcmofthegelisnotshownmMMnCI)ratherthaninHbufferToAilofannealedfragmentwasaddedMlofmMrCTP,,ulofH,and,ulofXMnbufferFivemicrocuriesofdried"aPdTTP(specificactivityapproximatelymCigumol)wasdissolvedinthisandunitDNApolymerase(Klenow)wasaddedIncubationwasformininiceOnemicroliterofMEDTAwasaddedbeforeloadingonamlSephadexGcolumnColumnbufferwasmMTris,pHmnMEDTAThelabeledfragmentwasfollowedbymonitor,collectedinaminimumvolume(approximatelyAtl),drieddown,andredissolvedin,lofXHbufferSamples(Al)ofthisweretakenfortreatmentasaboveFollowingthechasestep,AlofML>TG""Nw*~~~~~uATTCAAECqTIACTA>a,CACTAAl~~~~~AiAaCTGbewGTGAAACTGCFIGAutoradiographofanexperimentwithfragmentAasprimerontheviralstrandof,XDNAusingthesinglesiteribosubstitutionmethodElectrophoresiswasonagelatmAforhrThetopcmisnotshownInhibitorsusedwere(lefttoright)ddTTParaCTP,ddCTP,ddGTP,andddATPEDTAandAlofpancreaticribonucleaseAatmgmlwereaddedandincubatedforminatRESULTSFigsshowexamplesoftheuseofthemethodfordeterminingsequencesintheDNAofOXIntheexperimentshowninFigtwosmallrestrictionenzymefragments(AdandA,ref)wereusedasprimersonthecomplementarystrandandtherewasnofinaldigestionsteptocutbetweentheBiochemistrv:SangeretalI!,,::mwwnow"MkProcNatlAcadSciUSA()primerandthenewlysynthesizedDNAThisisthemostsimpleandrapidprocedure,requiringonlyapreliminaryannealingoftemplateandprimer,incubationofthefourseparatesampleswithDNApolymeraseandappropriatetriphosphates,followedbyachasewithunlabeleddATPandapplicationtothegelforelectrophoresisIntheseexperimentstheinhibitorsusedwereddGTP,ddATP,ddTTP,andaraCTPTheconditionsusedforthe"T"sampleswerenotentirelyoptimal,resultinginthefastermovingbandsbeingrelativelyweakThesequencescanbereadwithreasonableaccuracystartingatnucleotidesfromthe'endoftheprimerforaboutnucleotides(apartfromsomedifficultyatpositionwithAd)Forthenextnucleotidesthereissomeuncertaintyinthenumberofnucleotidesin"runs"becausebandsarenotactuallyresolvedWithlongerrestrictionenzymefragmentsasprimersitisnecessarytosplitthemofffromthenewlysynthesizedDNAchainsbeforetheelectrophoresisThisisnormallydonebydigestionwitharestrictionenzymeFigshowssuchanexperimentinwhichfragmentRwasusedasprimeronthecomplementarystrandofOXDNAInthisexperimentonlydideoxynucleosidetriphosphateswereusedasinhibitorsbecausetheresultswitharaCweremuchlesssatisfactorywhenarestrictionenzymewasusedforthesubsequentsplittingThismaybeduetothearaCbeingremovedbythe'exonucleaseactivityoftheDNApolymeraseduringtheincubationat(whichisnecessaryfortherestrictionenzymesplitting),resultinginafewCbandsbeingeitherveryfaintormissingAlternatively,theenzymemaybeabletoextendsomechainsbeyondthearaCatthehighertemperaturewhilebeingunabletodosoatlowertemperaturesaraATP,whichhasbeenusedonlyundertheseconditions,showsthesamelimitationsasaraCTPTheseproblemsdonotarisewhenddCTPisusedinthisreactionWithoneexception(positions,seebelow),asequenceofnucleotides,startingatapositionnucleotidesfromtherestrictionenzymesplittingsite,couldbereadoffthesequenceagreedwiththepublishedoneThisregionisbelievedtocontaintheoriginofviralstrandreplication(,)Thebandsbeyondpositionindicatedthattherewasanerrorintheprovisionalsequence(),andfurtherwork(tobepublishedlater)hasshownthatthetrinucleotideCGCshouldbeinsertedbetweenpositionsandWhenthistechniqueisusedtheproductsarecutwitharestrictionenzymeasabove,difficultiesariseifthereisasecondrestrictionenzymesiteclosetothefirstone,becausethiswillgiverisetoaseparatepatternofbandsthatissuperimposedonthenormalone,makinginterpretationimpossibleOnewayinwhichthiscanbeavoidedisbythesinglesiteribosubstitutionmethod(NLBrown,unpublished)AfterannealingofthetemplateandprimerasingleribonucleotideisincorporatedbyincubationwithDNApolymeraseinthepresenceofmanganeseandtheappropriateribonucleosidetriphosphateExtensionoftheprimeristhencarriedoutwiththeseparateinhibitorsasaboveandtheprimerissplitoffattheribonucleotidebyribonucleaseoralkaliThemethodisparticularlysuitableforusewithfragmentsobtainedwiththerestrictionenzymeAlu,whichsplitsatthetetranucleotidesequenceAGCTThisenzymeisinfactinhibitedbysinglestrandedDNAandcannotbeusedforthesubsequentsplittingoftheprimerfromthenewlysynthesizedDNAchainTheinitialincorporationiscarriedoutinthepresenceofrCTPandaPldTTPTheincorporationofthepfacilitatessubsequentpurificationontheSephadexcolumnFigshowsanexampleoftheuseofthismethodwithfragmentAontheviralstrandofOXDNAAsequenceofaboutnucleotidesstartingresiduesfromtheprimingsitecanbereadoffIntheprovisionalsequence()thisregionwasregardedasverytentativeMostofitisconfirmedbythisexperiment,butthereisaclearrevisionrequiredatpositionsThesequenceoftheviralstrandshouldreadATCAAC,replacingATTCACgivenintheprovisionalsequenceThereisdifficultyinreadingthesequenceat,wherethereisconsiderablevariationinthedistancebetweenbands,suggestingthepresenceofaloopedstructureFurtherworkinwhichtheelectrophoresiswascarriedoutatahighertemperatureindicatesthatthesequencehereisactuallyGCTCGCG(viralstrand)ie,aninsertionofCbetweenpositionsandintheprovisionalsequenceDISCUSSIONThemethoddescribedherehasanumberofadvantagesovertheplusandminusmethodsFirst,itissimplertoperformbecauseitrequiresnopreliminaryextension,thusavoidingoneincubationandpurificationonaSephadexcolumnItrequiresonlythecommerciallyavailableDNApolymeraseI(Klenowfragment)Theresultsappeartobemoreclearcutwithfewerartefactbands,andcanusuallybereadfurtherthanwiththeplusandminusmethodsIntermediatenucleotidesin"runs"showupasbands,thusavoidingasourceoferrorintheplusandminusmethodestimatingthenumberofnucleotidesinarunTheoreticallyonewouldexpectthedifferentbandsinaruntobeofthesamestrength,butthisisnotalwaysthecaseFrequently,thefirstnucleotideisthestrongest,butinthecaseofddCTPthesecondisthestrongest(seeFig)Thereasonsfortheseeffectsarenotunderstood,buttheydonotusuallycausedifficultieswithdeducingthesequencesForthelongersequencesinwhichtheseparatebandsinarunarenotresolved,experiencehasshownthatitisfrequentlypossibletoestimatethenumberofnucleotidesfromthestrengthandwidthofthebandTheinhibitormethodcanalsobeusedonasmallerscalethantheplusandminusmethodbecausebetterincorporationfromPlabeledtriphosphatesisobtainedThisispresumablyduetothelongerincubationperiodused,whichallowsamorequantitativeextensionofprimerchainsIngeneral,sequencesoffromtoaboutnucleotidesfromtheprimingsitecanbedeterminedwithreasonableaccuracyusingasingleprimerFrequentlyitispossibletoreadthegelsfurtherand,onoccasions,asequenceofaboutnucleotidesfromtheprimingsitehasbeendeterminedOccasionalartefactsareobserved,butthesecanusuallybereadilyidentifiedItseemslikelythattheseareusuallyduetocontaminantsinthefragmentsThemostseriousdifficultiesaredueto"pileups"ofbands,whichareusuallycausedbytheDNAformingbasepairedloopsundertheconditionsoftheacrylamidegelelectrophoresisThesepileupsareseenasanumberofbandsinthesamepositionorunusuallyclosetooneanotherontheelectrophoresisTheygenerallyoccuratdifferentpositionswhentheprimingiscarriedinoppositedirectionsalongtheDNAoverthesamesequenceAnexampleofthiseffectisseeninFigatposition,wherethereis

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