Transcription of DNA sequencing with chain-terminating inhibitors
1 , , ,December1977 BiochemistryDNAsequencingwithchain-termi natinginhibitors(DNApolymerase/nucleotid esequences/bacteriophage4X174) , , ,CambridgeCB22QH, ,October3, "plusandminus"method[Sanger, , (1975) ,441-4481butmakesuseofthe2',3'-dideoxyan darabinonu-cleosideanaloguesofthenormald eoxynucleosidetriphosphates, "plusandminus"method(1)isarelativelyrapi dandsimpletechniquethathasmadepossibleth edeterminationofthesequenceofthegenomeof bacteriophage4X174(2). ,neitherthe"plus"northe"minus"methodisco mpletelyaccurate,andinordertoestablishas e-quencebothmustbeusedtogether, ( ,inpress)hasrecentlydevelopedathirdmetho d,involvingribo-substi-tution,whichhasce rtainadvantagesovertheplusandminusmethod , (3), ,butitrequiresastrandseparationorequival entfrac-tionationofeachrestrictionenzyme fragmentstudied, , (4)showedthattheinhibitoryactivityof2',3 '-dideoxythymidinetriphosphate(ddTTP)onD NApolymeraseIdependsonitsbeingincorpo-ra tedintothegrowingoligonucleotidechainint heplaceofthymidylicacid(dT).]
2 BecausetheddTcontainsno3'-hydroxylgroup, thechaincannotbeextendedfurther, ,aswellastheotherthreedeoxyribonucleosid etriphosphates(oneofwhichislabeledwith32 p),amixtureoffragmentsallhavingthesame5' andwithddTresiduesatthe3' , '-hydroxylgroupisori-entedintranspositio nwithrespecttothe2' (ara)nucleotidesactaschainterminatingin- hibitorsofEscherichiacoliDNApolymeraseIi namannercomparabletoddT(4),althoughsynth esizedchainsendingin3'araCcanbefurtherex tendedbysomemammalianDNApolymerases(5).I nordertoobtainasuitablepatternofbandsfro mwhichanextensivesequencecan , (6,7), (8).Weessentiallyfollowedtheirprocedurea ndusedthemethodsofTener(9)andofHoardandO tt(10)toconvertittothetriphosphate,which wasthenpurifiedonDEAE-Sephadex, ; , ' (11).
3 Aftertosylationofthe3'-OHgroup(12)thecom poundwasconvertedtothe2',3'-didehydroder ivativewithsodiummethoxide(8).Theisobuty rylgroupwaspartlyre-movedduringthistreat mentandremovalwascompletedbyincubationin NH3( )overnightat45 .Thedidehydroderivativewasreducedtothedi deoxyderivative(8) ( ) '-O-monomethoxy-trityldeoxycytidine(Coll aborativeResearchInc.,Waltham,MA) :ThesymbolsC,T,A,andGareusedforthedeoxyr i-bonucleotidesinDNAsequences;theprefixd disusedforthe2',3'-dideoxyderivatives( ,ddATPis2',3'-dideoxyadenosine5'-tri-pho sphate); (1977)A14(-)A12d(-GATCGATC:..S:_<||..44 TIAxaAC,-t; ~~40320/t-'~(-AG-("QiTte4 Ozc20C11-TaG-A-2 (A4000T(,1Q(104-A-A---3999)(;001(oTI-A11 A--349800C39T0TA39601 ATAA3950Q( *.))))))))
4 'AAAIT35,10 ITATA3500-1AA3 UAs43)40jij~~~C.'GA;ATcAtA($48(-tit1-to3 430;jA7T-3-byC-CA3ox4810~~~~~~~( ~ (lefttoright)ddGTP,ddATP,ddTTP, (4) , ,Milwaukee, ,ugofOX174replicativeformin5,AlofH20wasm ixedwith1 Alofviralorcomplementarystrand4X174 DNA( )and1 MiofHX10buffer(13)andsealedinacapillaryt ube,heatedto1000for3min, "mix"and1ilofDNApolymerase(accordingtoKl enow,Boehringer,Mannheim)( ). ,1 MCiof[a-32P]dATP(specificactivityapproxi mately100mCi/,Mnwol) , , , , , , , , , , , , (TheconcentrationoftheddCTPwasuncertainb ecausetherewasinsufficientyieldtodetermi neit,buttherequireddilutionofthesolution wasdeterminedexperimentally.)))
5 , , , (chase)wasomittedsometermi-nationatAresi duesoccurredinallsamplesduetothelowconce ntrationofthe[ ,whereitwasunnecessarytocarryoutasubsequ entsplitting( ),thevariousreactionmixturesweredenature ddirectlyandappliedtotheacrylamidegelfor elec-trophoresis(1).Iffurthersplittingwa snecessary( ),1 Miloftheappropriaterestrictionenzymewasa ddedshortlyafterthedATP"chase, ( ,unpublished) "Mnbuffer"(66mMTrisCl, :' (1977)5465 GATCTaCddCGA~I~ *--G-GA-CGAAAA4340~~AACTG^cGAAG4330)* _CCCCC4320 BlitzAACC4-*-ATAG--I-~~~CAs;C4310=M~AA-_ WT'A4300*C~~~~~AATGTT'^ , (4units/0) ) ,2,ulofH20,and1, "a-32 PdTTP(specificactivityapproximately1mCi/ gumol)wasdissolvedinthisand1unitDNApolym erase(Klenow) , ,collectedinamini-mumvolume(approximatel y200 Atl),drieddown,andredis-solvedin30.]
6 (2Al) , >TG_""Nw*~~~~~ >a,CAC3550 TAA36l~~~~~ ; , (lefttoright) ,ddCTP,ddGTP, . (A12dandA14, ) !-,..-,::..--mww-141now" (1977) ,requiringonlyapreliminaryannealingoftem plateandprimer,incubationofthefoursepara tesampleswithDNApolymeraseandappropriate triphosphates, ,ddATP,ddTTP, "T"sampleswerenotentirelyoptimal, 'endoftheprimerforabout80nucleotides(apa rtfromsomedifficultyatposition3459withA1 2d).Forthenext50nucleotidesthereissomeun certaintyinthenumberofnucleotidesin"runs " '-exonucleaseactivityoftheDNApolymerased uringtheincubationat370(whichisnecessary fortherestrictionenzymesplitting), , ,whichhasbeenusedonlyunderthesecondition s, (positions4330-4343,seebelow),ase-quence of120nucleotides,startingataposition61nu cleotidesfromtherestrictionenzymesplitti ngsite,couldbereadoff; (2,14).
7 Thebandsbeyondposition4380indicatedthatt herewasanerrorintheprovisionalsequence(2 ),andfurtherwork(tobepub-lishedlater) ,difficultiesariseifthereisasecondrestri ctionenzymesiteclosetothefirstone,becaus ethiswillgiverisetoaseparatepatternofban dsthatissuperimposedonthenormalone, ( ,unpublished). , [ (2) , , ,wherethereisconsiderablevariationinthed istancebetweenbands, (viralstrand); , ,itissimplertoperformbe-causeitrequiresn opreliminaryextension, (Klenowfragment).Theresultsappeartobemor eclear-cutwithfewerartefactbands, "runs"showupasbands, , ,thefirstnucleotideisthestrongest,butint hecaseofddCTPthesecondisthestrongest( ).]
8 Thereasonsfortheseeffectsarenotunderstoo d, , , , ,onoccasions, , "pile-ups"ofbands, , , , '-O-monomethoxytrityldeoxyguanosine, , , (1975) , ,F.,Air, ,Barrell, ,Brown, ,Coulson, ,Fiddes, ,Hutchison, ,Slocombe, ,M.(1977)Nature265, , ,&Gilbert,W.(1977) !. , , ,Deutscher, ,Kornberg,A.,Russell, !. (1977)5467&Moffatt, (1969)Biochemistry8, , ,B.(1975) , , , (1969)Biochemistry8, ,K.(1974) , , ,Robins, ,Townsend, , (1966) , , (1961) , , , (1965) , , , (1972) , , ,McCarthy, , (1966)Biochem-istry5, , ,'Sanger, , (1976) , , (1976) ,UniversityofCam-bridge.
