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Proc.Nati. Acad. Sci. USA

Vol.74, No. 12, pp. 5463-5467,December1977 Biochemistry

DNA sequencing with chain-terminating inhibitors

(DNApolymerase/nucleotidesequences/bacteriophage4X174) F. SANGER, S. NICKLEN, AND A. R. COULSON

Medical Research Council Laboratory of Molecular Biology, CambridgeCB2 2QH,England ContributedbyF.Sanger,October 3, 1977

ABSTRACT Anewmethod for determining nucleotide se- quences in DNA is described. It is similar to the "plus and minus" method[Sanger,F.& Coulson, A. R. (1975) J.Mol. Biol.

94,441-4481 but makes use of the2',3'-dideoxy and arabinonu- cleosideanalogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.The technique has been applied to the DNA of bacteriophage4bX174and is more rapid and more accurate than either theplusor the minusmethod.

The "plus and minus" method (1) is a relatively rapid and simple technique that has made possible the determinationof the sequence of the genome ofbacteriophage 4X174 (2). It dependsontheuseofDNApolymerasetotranscribespecific regions of theDNAunder controlled conditions. Although the method is considerably more rapid and simple than other available techniques, neither the "plus" nor the "minus"

methodiscompletely accurate, andinorder toestablisha se- quence both mustbe used together, and sometimes confirma- tory dataarenecessary. W.M. Barnes(J. Mol.Biol.,inpress) hasrecently developed a third method, involving ribo-substi- tution,which has certain advantages over the plus andminus method, but this hasnotyet beenextensively exploited.

Anotherrapid and simple method thatdepends on specific chemicaldegradationoftheDNAhasrecently been described by Maxam and Gilbert (3), and this has also been usedexten- sivelyfor DNAsequencing. It hastheadvantageovertheplus and minusmethod thatitcanbeapplied todouble-stranded DNA, but it requires a strand separation or equivalent frac- tionationof eachrestrictionenzymefragmentstudied, which makesitsomewhat more laborious.

This paper describes a further method using DNA poly- merase,which makes use of inhibitors that terminate the newly synthesized chainsatspecific residues.

Principle of the Method. Atkinson et al. (4) showedthat the inhibitory activity of 2',3'-dideoxythymidine triphosphate (ddTTP)on DNApolymeraseIdependson itsbeing incorpo- rated into the growing oligonucleotide chain inthe placeof thymidylicacid(dT).Becausethe ddTcontains no3'-hydroxyl group,the chain cannot beextendedfurther,sothat termination occursspecificallyatpositions wheredT should beincorporated.

If aprimer andtemplateareincubatedwithDNApolymerase inthepresenceof a mixture of ddTTPanddTTP,aswellasthe other threedeoxyribonucleoside triphosphates (one ofwhich islabeled with32p),a mixtureof fragmentsall having the same 5'andwith ddT residues atthe3'endsisobtained. When this mixture is fractionated by electrophoresis on denaturing acrylamide gels the pattern of bands showsthedistributionof dTs in the newlysynthesized DNA. By usinganalogouster- minatorsfor theother nucleotidesinseparate incubations and running thesamplesinparallelonthegel, apatternof bands isobtained from which the sequencecan bereadoff as in the otherrapid techniques mentioned above.

Twotypesofterminatingtriphosphateshave beenused-the dideoxy derivatives and the arabinonucleosides. Arabinoseis

5463

a stereoisomerofribose in which the 3'-hydroxyl group is ori- ented in trans position with respect to the 2'-hydroxyl group.

The arabinosyl (ara) nucleotides act as chain terminatingin- hibitors ofEscherichia coli DNA polymerase Iin amanner comparable to ddT (4), although synthesized chains endingin 3'araC can be further extended by some mammalianDNA polymerases (5).Inorder to obtain a suitable pattern of bands from which an extensivesequence can be read it is necessary tohavea ratioof terminatingtriphosphate to normal triphos- phate such that only partial incorporation of the terminator occurs. Forthedideoxy derivatives this ratio is about 100,and for thearabinosyl derivatives about5000.

METHODS

Preparation of theTriphosphate Analogues. The prepa- rationof ddTTP has been described (6, 7), and the materialis now commercially available. ddA has been prepared by McCarthyetal. (8).Weessentiallyfollowed their procedure andused the methods ofTener(9) and of Hoard andOtt(10) to convert it tothetriphosphate, which wasthenpurifiedon DEAE-Sephadex,using a 0.1-1.0Mgradientoftriethylamine carbonateatpH 8.4. Thepreparationof ddGTPand ddCTP has not been described previously; however we applied the samemethodasthatused forddATPandobtained solutions having therequisiteterminating activities.Theyieldswerevery low and this can hardly be regarded as adequate chemical characterization. However,therecanbe little doubt that the activity wasduetothedideoxy derivatives.

Thestarting materialfor theddGTPwas N-isobutyryl-5'- O-monomethoxytrityldeoxyguanosine prepared by F. E.

Baralle (11). After tosylation of the 3'-OH group (12) the compoundwasconvertedtothe2',3'-didehydroderivativewith sodium methoxide (8). The isobutyryl group was partly re- movedduring thistreatmentand removal wascompleted by incubationinNH3 (specificgravity0.88)overnightat45°.The didehydroderivativewasreducedtothedideoxy derivative (8) and convertedtothetriphosphateasfor the ddATP. Themo- nophosphate was purified by fractionation on a DEAE-Se- phadex column using a triethylamine carbonate gradient (0.025-0.3 M) but thetriphosphatewas notpurified.

ddCTPwaspreparedfromN-anisoyl-5'-O-monomethoxy- trityldeoxycytidine (Collaborative Research Inc., Waltham, MA) by the above method but the final purification on DEAE-Sephadexwasomitted because theyieldwasverylow and the solution containedtherequiredactivity.The solution wasuseddirectlyintheexperimentsdescribedinthispaper.

An attemptwasmade toprepare the triphosphate of the intermediate didehydrodideoxycytidinebecause Atkinsonet Abbreviations:ThesymbolsC, T, A,andGareusedfor thedeoxyri- bonucleotidesin DNAsequences; theprefixddisused for the2',3'-

dideoxy derivatives (e.g., ddATP is 2',3'-dideoxyadenosine 5'-tri-

phosphate);theprefixara isused for the arabinoseanalogues.

(2)

Proc. Natl.Acad. Sci. USA74(1977)

A14 (-) A12d(-

G A T C G A T C

:.. S: _

<

| ..

|

44

T I AxaA C,-t;ri.

4040 I A GAC~ ~

40320 /t -'~ (-AG-(" QiT t e

4Ozc20C1 1-Ta G-A- 2:e.

401A0A1

(A

4000 T(,1 Q ( 1 04-A-A-- -

3999)(;001(oT I-1A 1 A- -

349800C

39T0

T A 39601AT

A A

3950Q(

o A 3940 GrT

3930 T A

_ ..

0*..

_

OFO

4.m.

P-t..A'AAAIT

35,10

IT ATA 3500

-1

A

UAs4

A3

3)40

jij~~~C.'G A; AT c At

A

( $48(

-t it1-to3430

;jA 7T-3- by C

-C A 3ox4810

~~~~~~~( AC 347

A 3460

A

_ Ad GAT 34150 ACs. iAA

~ACTGA

-CA 3440

A AA 3

A

AC C 34300

C A A 3420

FIG. 1. Autoradiographoftheacrylamide gel fromthe sequence determination using restriction fragmentsA12d andA14 as primers on the complementary strand of IX174DNA.The inhibitorsused were(lefttoright)ddGTP,ddATP,ddTTP, and araCTP.Electrophoresiswason a12%6acrylamide gelat40mA for14hr.Thetop 10 cmofthegelis not shown. TheDNAsequenceis written fromlefttorightandupwardsbeside thecorrespondingbandsontheradioautograph.Thenumberingis as given in ref. 2.

al. (4)have shown that thedidehydrodideoxy-TTPisalso active as a terminator. However, wewereunsuccessfulinthis. These compounds seem much less stable than the dideoxy deriva- tives.

araATPand araCTPwereobtained from P-LBiochemicals Inc., Milwaukee, WI.

SequencingProcedure. Restriction enzyme fragments were obtained from

OX174

replicative form and separated by elec- trophoresisonacrylamide gels. The material obtained from5 ,ugof

OX174

replicativeform in 5,Alof H20 was mixed with 1

Al

of viralorcomplementary strand4X174DNA (0.6

jg)

and 1 MiofHX10buffer(13) and sealed in a capillary tube, heated to1000 for3min,and then incubated at670 for 30 min. The solution wasdiluted to 20 Al with H buffer and 2

Al

samples weretakenfor each incubation and mixed with 2Ml of the ap- propriate "mix"and 1

il

of DNA polymerase (according to Klenow, Boehringer, Mannheim) (0.2 units). Each mix con- tained 1.5XHbuffer,1 MCiof [a-32P]dATP(specific activity approximately 100

mCi/,Mnwol)

and the following other tri- phosphates.

ddT: 0.1 mMdGTP, 0.1 mMdCTP, 0.005 mM dTTP, 0.5 mMddTTP

ddA: 0.1 mMdGTP, 0.1 mMdCTP, 0.1 mM dTTP, 0.5 mM ddATP

ddG: 0.1 mMdCTP, 0.1 mMdTTP, 0.005 mM dGTP, 0.5 mMddGTP

ddC: 0.1 mMdGTP,0.1 mMdTTP, 0.005mMdCTP, approximately0.25mMddCTP

(Theconcentrationof theddCTPwasuncertain becausethere wasinsufficient yield to determine it, but the required dilution of the solutionwasdetermined experimentally.)

araC: 0.1 mMdGTP,0.1 mMdTTP, 0.005mMdCTP, 12.5 mM araCTP

Incubation was at room temperature for 15 min. Then 1Ml of0.5 mMdATPwasadded and incubationwascontinuedfor afurther15min. Ifthis step(chase) wasomittedsome termi- nation at A residues occurred in all samples due to the low concentrationofthe[a-32PldATP. Withsmallprimers,where it wasunnecessarytocarryoutasubsequentsplitting (as in the experimentshowninFig. 1), the various reaction mixtureswere denatureddirectlyandappliedtotheacrylamide gelforelec- trophoresis(1). If further splitting was necessary (see Fig. 2), 1

Mil

of the appropriate restriction enzyme wasadded shortly after the dATP"chase, and incubation was at 370.

The single-site ribo-substitution procedure (N. L. Brown, unpublished) was carried out as follows. The annealing of template and primer was carried out as above but in "Mn buffer" (66 mMTrisCl, pH7.4/1.5mM

2-mercaptoethanol/

5464

Biochemistry:' Sanger

etal.

(3)

Proc.Nati. Acad. Sci. USA 74(1977) 5465

G

A T C T

aC

ddC

G

A

~I~A9TiTTC

4380

Si

E

CGTT -CA7

- 4370

ohs

PAGACAGA

4360 ---AC--A A 4340

CGAG 4330

ACCA

4320-6-.

T A 4350

- - TG T T T

= * - -G-GA-CGAAAA 4340

~~ AACTG

^cGAAG 4330)

_

CCCCC 4320

BlitzACC A 4

-*- ATAG

- -I-~ ~~CAs;C 431 0

= M~AA

-_W T ' A 4300

* C~~~~~AAT G

T T

'

^cA

4290

cA

A

A - C4270

G0 ATT

T 4260

FIG. 2. Autoradiograph from an experiment using fragment R4 asprimeronthecomplementarystrandofpX174DNA.Condi- tions were as inFig. 1 with thefollowingexceptions:ddCTPwasused asinhibitor insteadofaraCTP. After incubationofthe solutionsat roomtemperaturefor 15min,1 glof0.5mMdATP and1glofre- strictionenzyme Hae III(4units/0)wereaddedand thesolutions wereincubatedat370for 10 min.TheHae III cutsclosetotheHindII

siteandit wasusedbecause itwasmorereadilyavailable. The elec- trophoresiswasona 12%acrylamide gelat40mA for 14 hr. The top 10.5cmof thegelis notshown.

0.67 mM MnCI2) ratherthanin Hbuffer. To 7Ailofannealed fragmentwasadded 1 Mlof10mMrCTP,2,ulofH20,and 1 ,ul of 10 XMnbuffer. Five microcuriesof dried"a-32PdTTP (specificactivityapproximately 1 mCi/gumol)wasdissolvedin

thisand 1 unit DNA polymerase (Klenow) wasadded. Incu- bationwasfor30mininice.Onemicroliter of0.2 M EDTA was added beforeloadingona 1-mlSephadexG-100column.Col- umnbuffer was 5mM Tris, pH7.5/0.1 mnM EDTA. Thela- beled fragmentwasfollowedby monitor,collectedinamini- mum volume(approximately 200

Atl),

drieddown,andredis- solvedin30,.lof 1 XHbuffer. Samples(2Al)of thisweretaken fortreatmentasabove.Followingthe chasestep, 1Alof0.1 M

- -. _ L>TG

_ ""Nw*~~~~~u -ATT C

..-

AA

EC

3520-

q - - - -ACTATI---3530

>a , C AC 3550

T AA36

l~~~~~

AiA

.-34

a- C

3480 3490 3500 3510

- - -T 1- G 3570

-bew -;---G

TG

-- AA

----A 3580

C T 3590 G

C

FIG. 3. Autoradiograph of anexperiment with fragmentA8 as primer on theviral strand of ,X174DNAusing thesingle-siteribo- substitution method.Electrophoresiswas ona12%gelat40mA for 6hr.Thetop 5.5cmisnotshown.Inhibitorsusedwere(lefttoright) ddTTP.araCTP, ddCTP, ddGTP,andddATP.

EDTAand1

Al

of pancreaticribonucleaseAat 10mg/mlwere added and incubated for60minat37°.

RESULTS

Figs. 1-3show examplesof theuse ofthe method for deter- mining sequences in the DNA of OX174. In the experiment showninFig. 1twosmallrestrictionenzymefragments

(A12d

and A14, ref. 2)wereusedasprimersonthecomplementary strandand therewas nofinaldigestionsteptocutbetweenthe Biochemistrv:

Sanger

etal.

I

!-,..-,::.. --mww-141now

"Mk

(4)

Proc.Natl.Acad. Sci. USA 74(1977)

primerand the newlysynthesized DNA.Thisisthemostsimple andrapid procedure, requiringonlyapreliminary annealing oftemplateandprimer, incubation of the fourseparatesamples with DNApolymerase andappropriatetriphosphates, followed byachase with unlabeled dATPand applicationtothe gel for electrophoresis. Intheseexperimentstheinhibitorsusedwere ddGTP, ddATP, ddTTP, andaraCTP. Theconditionsused for the "T" samples were not entirely optimal, resulting in the faster-moving bands being relatively weak.

Thesequencescanbe read with reasonableaccuracystarting at 88nucleotides fromthe5'end of theprimer forabout 80

nucleotides (apartfromsomedifficulty atposition3459with A12d). For thenext50nucleotides thereis someuncertainty inthe number ofnucleotidesin"runs" because bandsarenot

actually resolved.

With longer restriction enzyme fragments as primers it is

necessary tosplit them off from the newlysynthesized DNA chains before theelectrophoresis. Thisisnormally done by di- gestionwitharestrictionenzyme. Fig. 2shows suchanexper-

iment inwhich fragmentR4 wasusedasprimeronthecom-

plementary strand of OX174 DNA. In this experiment only dideoxynucleoside triphosphateswereusedasinhibitors because the results witharaC were much less satisfactory whena re-

strictionenzyme wasusedfor the subsequent splitting. Thismay be duetothearaCbeing removed by the3'-exonucleaseactivity

of the DNApolymeraseduring the incubation at370 (which isnecessaryfortherestrictionenzymesplitting), resulting in

afewCbandsbeing eitherveryfaintormissing.Alternatively, theenzyme maybe abletoextendsomechainsbeyond thearaC atthehighertemperature while beingunabletodosoatlower

temperatures. araATP, which has been used only under these conditions, shows the same limitations as araCTP. These problems donotarisewhen ddCTPisusedinthisreaction.

Withoneexception(positions4330-4343, seebelow), a se- quenceof120nucleotides,startingataposition61 nucleotides fromtherestrictionenzymesplittingsite,could be read off; the

sequenceagreed with thepublishedone. Thisregionisbelieved

tocontain theorigin of viral strand replication (2, 14). The bandsbeyondposition4380indicated that therewas anerror

intheprovisionalsequence(2),and further work(tobepub- lished later) has shown that the trinucleotide C-G-C shouldbe inserted betweenpositions4380and4381.

When this technique isusedtheproductsarecutwithare-

strictionenzyme asabove, difficultiesariseifthereisasecond restrictionenzymesiteclose tothe firstone, becausethis will give risetoaseparate patternof bands thatissuperimposedon

thenormalone,makinginterpretation impossible.Onewayin

which thiscanbe avoidedisbythe single-siteribo-substitution method (N. L. Brown, unpublished). After annealing of the template andprimerasingleribonucleotideisincorporated by incubation withDNApolymeraseinthepresenceofmanganese andtheappropriateribonucleoside triphosphate. Extensionof the primer isthen carriedout with theseparateinhibitors as

above and theprimerissplit offatthe ribonucleotide by ribo- nucleaseoralkali. The methodisparticularly suitableforuse with fragments obtained with the restriction enzyme Alu, which splits at the tetranucleotide sequence A-G-C-T. This

enzymeisinfactinhibitedbysingle-strandedDNAandcannot

be used for thesubsequent splitting of the primer from the newly synthesized DNA chain. The initial incorporation is

carriedoutin thepresenceofrCTPand[a-32PldTTP. Thein-

corporationof the32pfacilitates subsequent purificationonthe Sephadexcolumn.

Fig. 3 shows an example of the use of this method with fragmentA8ontheviral strand ofOX174 DNA. Asequence

of about110 nucleotides starting33residues from thepriming site can bereadoff. Intheprovisionalsequence (2) thisregion wasregardedas verytentative. Most of it is confirmed by this experiment, but there is a clear revision required at positions 3524-3530. Thesequence of the viral strand should read A- T-C-A-A-C, replacingA-T-T-C- --A-C given in theprovisional sequence. There is difficulty in reading the sequence at 3543-3550, where there is considerable variation in the distance between bands,suggestingthepresence of a loopedstructure.

Further work in which the electrophoresis was carried out at ahighertemperatureindicates that the sequence here is actu- allyG-C-T-C-G-C-G (viral strand); i.e., an insertion of C be- tween positions 3547 and 3548 in theprovisional sequence.

DISCUSSION

Themethoddescribed here has a number of advantages over the plus andminus methods. First,it is simpler to perform be- cause it requires no preliminary extension, thus avoiding one incubationandpurificationon a Sephadexcolumn. Itrequires only thecommercially available DNA polymerase I (Klenow fragment). Theresults appear to be more clear-cut with fewer artefact bands, and can usually be read further than with the plus and minusmethods. Intermediate nucleotides in "runs"

show up asbands, thusavoidingasource of error in the plus and minusmethod-estimatingthenumber of nucleotides in a run.

Theoretically onewouldexpectthedifferentbands in a run to be ofthe same strength, but this is not always the case. Fre- quently, the firstnucleotideisthe strongest, but in the case of ddCTPthesecondisthestrongest (seeFig. 2). Thereasonsfor these effectsare notunderstood,buttheydo notusually cause difficulties with deducing the sequences. For the longer se- quences in which theseparatebands in a run are notresolved, experiencehasshown that it isfrequently possibletoestimate thenumber ofnucleotidesfrom thestrength and width of the band.

Theinhibitormethod can also be used on a smaller scale than the plus andminus method because betterincorporation from 32P-labeled triphosphatesisobtained. This ispresumablydue to the longer incubation period used, which allows a more quantitative extensionofprimer chains.

Ingeneral, sequences of from 15 toabout 200 nucleotides fromtheprimingsite can bedeterminedwithreasonableac- curacyusing asingleprimer. Frequentlyit ispossibletoread the gels further and, on occasions, a sequence ofabout 300 nucleotides from thepriming site has beendetermined. Oc- casionalartefactsareobserved,butthesecanusuallybereadily identified. It seems likely that these are usually due to con- taminants in thefragments. The most seriousdifficulties are due to "pile-ups" ofbands, which are usually caused by the DNA forming base-paired loops under theconditions of the acrylamide gel electrophoresis. These pile-ups are seen as a number of bands in the samepositionorunusuallyclose to one another on the electrophoresis. They generally occur at dif- ferent positions when theprimingiscarriedinopposite direc- tions along the DNA over the samesequence. Anexample of this effect is seen in Fig. 2 at position 4330, where there is a single strong bandin theGchannelthat in factrepresents four G residues. They are presumably forming a stable loop by pairing with the four Cs atpositions 4323-4326. Another ex- ampleis in Fig. 3 atpositions 3545-3550. Thiseffectislikely to be found in all the rapid techniques that use gel electro- phoresis.

Itisfelt that for anaccuratedeterminationofsequenceone should notrely completely on single results obtained bythis methodalone but thatconfirmationshould beobtainedbysome 5466

Biochemistry: Sanger

etal.

(5)

Biochemistry: Sangeretal.

other techniqueor bypriming onthe opposite strand. This consideration probably appliestoall other available methods also. Themaindisadvantage of thepresentmethodisthe dif- ficulty in obtaining all the inhibitors-particularly ddGTP, whichisnotcommercially available.

We wishtothank Dr. K. Geider foragift of ddTTP, Dr. F. E. Baralle foragift of N-isobutyryl-5'-O-monomethoxytrityldeoxyguanosine, and Dr. M.J. Gait for useful adviceonthesynthetic work.

1. Sanger, F. &Coulson, A. R. (1975)J.Mol.Blol. 94,441-448.

2. Sanger, F., Air,G. M., Barrell, B.G.,Brown, N. L.,Coulson,A.

R., Fiddes, J. C., Hutchison, C.A.,Slocombe, P. M.&Smith,M.

(1977) Nature265,687-695.

3. Maxam, A. M,&Gilbert, W. (1977) Proc. Nat!. Acad. Sci. USA 74,560-564.

4. Atkinson, M. R., Deutscher,M. P.,Kornberg,A.,Russell,A.F.

Proc. Nat!. Acad. Sci. USA 74 (1977) 5467

&Moffatt,J.G.(1969)Biochemistry8, 4897-4904.

5. Hunter, T. &Francke,B.(1975)J.Virol. 15,759-775.

6. Russell, A. F. & Moffatt,J. G. (1969)Biochemistry 8, 4889- 4896.

7. Geider,K.(1974)Eur.J.Biochem. 27,555-563.

8. McCarthy,J.R.,Robins,M.J.,Townsend,L. B. &Robins,R. K.

(1966)J. Am.Chem.Soc. 88, 1549-1553.

9. Tener, G. M. (1961)J. Am.Chem.Soc. 83, 159-168.

10. Hoard,D. E. &Ott, D. G. (1965)J. Am.Chem.Soc.87,1785- 1788.

11. Buchi,H. &Khorana,H.G.(1972)J.Mol.Biol. 72,251-288.

12. Robins,M.J.,McCarthy,J. R. &Robins,R. K.(1966)Biochem- istry5,224-231.

13. Air,G. M.,'Sanger, F. &Coulson,A. R.(1976)J.Mol.Biol. 108, 519-533.

14. Slocombe,P. M.(1976) Ph.D.Dissertation, UniversityofCam- bridge.

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