Proc. Nati. Acad. Sci. USA
`Vol. 74, No. 12, pp. 5463-5467, December 1977
`Biochemistry
`
`DNA sequencing with chain-terminating inhibitors
`(DNA polymerase/nucleotide sequences/bacteriophage 4X174)
`F. SANGER, S. NICKLEN, AND A. R. COULSON
`Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England
`Contributed by F. Sanger, October 3, 1977
`
`A new method for determining nucleotide se-
`ABSTRACT
`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 the 2',3'-dideoxy and arabinonu-
`cleoside analogues of the normal deoxynucleoside triphosphates,
`which act as specific chain-terminating inhibitors of DNA
`polymerase. The technique has been applied to the DNA of
`bacteriophage 4bX174 and is more rapid and more accurate than
`either the plus or the minus method.
`The "plus and minus" method (1) is a relatively rapid and
`simple technique that has made possible the determination of
`the sequence of the genome of bacteriophage 4X174 (2). It
`depends on the use of DNA polymerase to transcribe specific
`regions of the DNA under controlled conditions. Although the
`method is considerably more rapid and simple than other
`available techniques, neither the "plus" nor the "minus"
`method is completely accurate, and in order to establish a se-
`quence both must be used together, and sometimes confirma-
`tory data are necessary. W. M. Barnes (J. Mol. Biol., in press)
`has recently developed a third method, involving ribo-substi-
`tution, which has certain advantages over the plus and minus
`method, but this has not yet been extensively exploited.
`Another rapid and simple method that depends on specific
`chemical degradation of the DNA has recently been described
`by Maxam and Gilbert (3), and this has also been used exten-
`sively for DNA sequencing. It has the advantage over the plus
`and minus method that it can be applied to double-stranded
`DNA, but it requires a strand separation or equivalent frac-
`tionation of each restriction enzyme fragment studied, which
`makes it somewhat more laborious.
`This paper describes a further method using DNA poly-
`merase, which makes use of inhibitors that terminate the newly
`synthesized chains at specific residues.
`Principle of the Method. Atkinson et al. (4) showed that the
`inhibitory activity of 2',3'-dideoxythymidine triphosphate
`(ddTTP) on DNA polymerase I depends on its being incorpo-
`rated into the growing oligonucleotide chain in the place of
`thymidylic acid (dT). Because the ddT contains no 3'-hydroxyl
`group, the chain cannot be extended further, so that termination
`occurs specifically at positions where dT should be incorporated.
`If a primer and template are incubated with DNA polymerase
`in the presence of a mixture of ddTTP and dTTP, as well as the
`other three deoxyribonucleoside triphosphates (one of which
`is labeled with 32p), a mixture of fragments all having the same
`5' and with ddT residues at the 3' ends is obtained. When this
`mixture is fractionated by electrophoresis on denaturing
`acrylamide gels the pattern of bands shows the distribution of
`dTs in the newly synthesized DNA. By using analogous ter-
`minators for the other nucleotides in separate incubations and
`running the samples in parallel on the gel, a pattern of bands
`is obtained from which the sequence can be read off as in the
`other rapid techniques mentioned above.
`Two types of terminating triphosphates have been used-the
`dideoxy derivatives and the arabinonucleosides. Arabinose is
`
`a stereoisomer of ribose 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 terminating in-
`hibitors of Escherichia coli DNA polymerase I in a manner
`comparable to ddT (4), although synthesized chains ending in
`3' araC can be further extended by some mammalian DNA
`polymerases (5). In order to obtain a suitable pattern of bands
`from which an extensive sequence can be read it is necessary
`to have a ratio of terminating triphosphate to normal triphos-
`phate such that only partial incorporation of the terminator
`occurs. For the dideoxy derivatives this ratio is about 100, and
`for the arabinosyl derivatives about 5000.
`
`METHODS
`Preparation of the Triphosphate Analogues. The prepa-
`ration of ddTTP has been described (6, 7), and the material is
`now commercially available. ddA has been prepared by
`McCarthy et al. (8). We essentially followed their procedure
`and used the methods of Tener (9) and of Hoard and Ott (10)
`to convert it to the triphosphate, which was then purified on
`DEAE-Sephadex, using a 0.1-1.0 M gradient of triethylamine
`carbonate at pH 8.4. The preparation of ddGTP and ddCTP
`has not been described previously; however we applied the
`same method as that used for ddATP and obtained solutions
`having the requisite terminating activities. The yields were very
`low and this can hardly be regarded as adequate chemical
`characterization. However, there can be little doubt that the
`activity was due to the dideoxy derivatives.
`The starting material for the ddGTP was N-isobutyryl-5'-
`O-monomethoxytrityldeoxyguanosine prepared by F.
`E.
`Baralle (11). After tosylation of the 3'-OH group (12) the
`compound was converted to the 2',3'-didehydro derivative with
`sodium methoxide (8). The isobutyryl group was partly re-
`moved during this treatment and removal was completed by
`incubation in NH3 (specific gravity 0.88) overnight at 45°. The
`didehydro derivative was reduced to the dideoxy derivative (8)
`and converted to the triphosphate as for the ddATP. The mo-
`nophosphate was purified by fractionation on a DEAE-Se-
`phadex column using a triethylamine carbonate gradient
`(0.025-0.3 M) but the triphosphate was not purified.
`ddCTP was prepared from N-anisoyl-5'-O-monomethoxy-
`trityldeoxycytidine (Collaborative Research Inc., Waltham,
`MA) by the above method but the final purification on
`DEAE-Sephadex was omitted because the yield was very low
`and the solution contained the required activity. The solution
`was used directly in the experiments described in this paper.
`An attempt was made to prepare the triphosphate of the
`intermediate didehydrodideoxycytidine because Atkinson et
`
`Abbreviations: The symbols C, T, A, and G are used for the deoxyri-
`bonucleotides in DNA sequences; the prefix dd is used for the 2',3'-
`dideoxy derivatives (e.g., ddATP is 2',3'-dideoxyadenosine 5'-tri-
`phosphate); the prefix ara is used for the arabinose analogues.
`
`5463
`
`PGDX EX. 1016
`Page 1 of 5
`
`

`

`Proc. Natl. Acad. Sci. USA 74 (1977)
`
`A14 (-)
`
`A 12d (-
`
`G
`
`A T C G A T
`
`C
`
`_
`
`_
`0*..
`
`..
`
`_
`
`OFO
`
`P-t..A'AAAIT 35,10
`IT AT A 3500
`-1
`A A3UAs43)40
`jij~~~C.'GA;AT
`$48(
`At A (
`c
`7T-3- by
`; jA
`
`C
`
`3ox4810
`-C A
`~~~~~~~( AC 3 4 7
`-t it1-to3430
`
`~ACTGA 3440-CA
`
`5464
`
`Biochemistry:' Sanger et al.
`
`44 T I AxaA
`
`C,-t;ri.
`
`A
`
`<||..
`
`:.. S:
`
`-
`
`A GAC~ ~
`4040 I
`40320 /t -'~ (-AG-(" QiT
`e
`t
`1-Ta G-A- 2:e.
`401A0A1(A
`4Ozc20C1
`1 04-A-A--
`4000 T(,1
`Q
`(
`3999)(;001(oT
`1 A- -
`I- A1
`349800C
`39601AT
`39T0 T
`
`A
`
`A
`3950Q(
`o A
`3940 GrT
`
`3930 T A
`
`4.m.
`
`A
`
`3460
`
`iAA
`
`ACs.
`A
`_ Ad GAT 34150
`
`AC C 34 300
`A
`
`C
`
`A
`
`A A 3420
`AA 3
`
`Autoradiograph of the acrylamide gel from the sequence determination using restriction fragments A12d and A14 as primers on the
`FIG. 1.
`complementary strand of IX174 DNA. The inhibitors used were (left to right) ddGTP, ddATP, ddTTP, and araCTP. Electrophoresis was on
`a 12%6 acrylamide gel at 40 mA for 14 hr. The top 10 cm of the gel is not shown. The DNA sequence is written from left to right and upwards beside
`the corresponding bands on the radioautograph. The numbering is as given in ref. 2.
`
`al. (4) have shown that the didehydrodideoxy-TTP is also active
`as a terminator. However, we were unsuccessful in this. These
`compounds seem much less stable than the dideoxy deriva-
`tives.
`araATP and araCTP were obtained from P-L Biochemicals
`Inc., Milwaukee, WI.
`Sequencing Procedure. Restriction enzyme fragments were
`obtained from OX174 replicative form and separated by elec-
`trophoresis on acrylamide gels. The material obtained from 5
`,ug of OX174 replicative form in 5 ,Al of H20 was mixed with
`1 Al of viral or complementary strand 4X174 DNA (0.6 jg) and
`1 Mi of H X 10 buffer (13) and sealed in a capillary tube, heated
`to 1000 for 3 min, and then incubated at 670 for 30 min. The
`solution was diluted to 20 Al with H buffer and 2 Al samples
`were taken for each incubation and mixed with 2 Ml of the ap-
`propriate "mix" and 1 il of DNA polymerase (according to
`Klenow, Boehringer, Mannheim) (0.2 units). Each mix con-
`tained 1.5 X H buffer, 1 MCi of [a -32P]dATP (specific activity
`approximately 100 mCi/,Mnwol) and the following other tri-
`phosphates.
`ddT: 0.1 mM dGTP, 0.1 mM dCTP, 0.005 mM dTTP, 0.5
`mM ddTTP
`ddA: 0.1 mM dGTP, 0.1 mM dCTP, 0.1 mM dTTP, 0.5 mM
`ddATP
`
`ddG: 0.1 mM dCTP, 0.1 mM dTTP, 0.005 mM dGTP, 0.5
`mM ddGTP
`ddC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP,
`approximately 0.25 mM ddCTP
`(The concentration of the ddCTP was uncertain because there
`was insufficient yield to determine it, but the required dilution
`of the solution was determined experimentally.)
`araC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP, 12.5
`mM araCTP
`Incubation was at room temperature for 15 min. Then 1 Ml
`of 0.5 mM dATP was added and incubation was continued for
`a further 15 min. If this step (chase) was omitted some termi-
`nation at A residues occurred in all samples due to the low
`concentration of the [a-32PldATP. With small primers, where
`it was unnecessary to carry out a subsequent splitting (as in the
`experiment shown in Fig. 1), the various reaction mixtures were
`denatured directly and applied to the acrylamide gel for elec-
`trophoresis (1). If further splitting was necessary (see Fig. 2),
`1 Mil of the appropriate restriction enzyme was added 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 mM TrisCl, pH 7.4/1.5 mM 2-mercaptoethanol/
`
`PGDX EX. 1016
`Page 2 of 5
`
`

`

`=M~AA
`
`Biochemistrv: Sanger et al.
`
`G A T C
`
`-_W
`
`*
`
`~I~A9TiTTC 4380
`Si E
`-CGTT -CA7 4370
`ohsPAGACAGA 4360
`CGAG 4330
`ACCA 4320-6-.T A 4350
`- - TG T T T
`------AC--A A 4340
`= * - -G-GA-C GAAAA 4340
`^cGAAG 4330)
`*¢_ CCCCC 4320
`ACC
`4
`Blitz A
`AT AG
`-*-
`- -I-~ ~~CAs;C 431 0
`AACTG
`~~
`T '
`A 4300
`C ~~~~~AAT
`G
`
`T
`
`'
`
`T
`^cA 4290
`
`c A
`
`A
`
`A
`
`- C4270
`
`G0
`
`TT
`
`A
`
`T 4260
`
`Autoradiograph from an experiment using fragment
`FIG. 2.
`R4 as primer on the complementary strand of pX174 DNA. Condi-
`tions were as in Fig. 1 with the following exceptions: ddCTP was used
`as inhibitor instead of araCTP. After incubation of the solutions at
`room temperature for 15 min, 1 gl of 0.5 mM dATP and 1 gl of re-
`striction enzyme Hae III (4 units/0) were added and the solutions
`were incubated at 370 for 10 min. The Hae III cuts close to the HindII
`site and it was used because it was more readily available. The elec-
`trophoresis was on a 12% acrylamide gel at 40 mA for 14 hr. The top
`10.5 cm of the gel is not shown.
`
`0.67 mM MnCI2) rather than in H buffer. To 7 Ail of annealed
`fragment was added 1 Ml of 10 mM rCTP, 2 ,ul of H20, and 1
`,ul of 10 X Mn buffer. Five microcuries of dried "a-32P dTTP
`(specific activity approximately 1 mCi/gumol) was dissolved in
`this and 1 unit DNA polymerase (Klenow) was added. Incu-
`bation was for 30 min in ice. One microliter of 0.2 M EDTA was
`added before loading on a 1-ml Sephadex G-100 column. Col-
`umn buffer was 5 mM Tris, pH 7.5/0.1 mnM EDTA. The la-
`beled fragment was followed by monitor, collected in a mini-
`mum volume (approximately 200 Atl), dried down, and redis-
`solved in 30 ,.l of 1 X H buffer. Samples (2 Al) of this were taken
`for treatment as above. Following the chase step, 1 Al of 0.1 M
`
`..-
`
`Proc. Nati. Acad. Sci. USA 74 (1977)
`
`5465
`
`T aC ddC G A
`
`- -.
`
`_
`
`L>TG
`-A T T C
`
`_ ""Nw*~~~~~u
`
`3480
`3490
`3500
`3510
`
`q
`l~~~~~
`-
`-
`
`I
`
`AA EC3520-
`TI
`----- 3530
`ACTA
`
`-
`
`-
`
`>a ,
`
`T
`C
`
`AA36
`AC 3550
`.-34
`AiA
`
`--mww -141now
`
`!-,..-,::..
`"Mk
`
`-
`
`-
`
`-bew
`
`a- C
`-T 1- G 3570
`-;---GTG
`
`-- AA
`----A 3580
`
`C T 3590
`
`G
`
`C
`
`Autoradiograph of an experiment with fragment A8 as
`FIG. 3.
`primer on the viral strand of ,X174 DNA using the single-site ribo-
`substitution method. Electrophoresis was on a 12% gel at 40 mA for
`6 hr. The top 5.5 cm is not shown. Inhibitors used were (left to right)
`ddTTP. araCTP, ddCTP, ddGTP, and ddATP.
`EDTA and 1 Al of pancreatic ribonuclease A at 10 mg/ml were
`added and incubated for 60 min at 37°.
`
`RESULTS
`Figs. 1-3 show examples of the use of the method for deter-
`mining sequences in the DNA of OX174. In the experiment
`shown in Fig. 1 two small restriction enzyme fragments (A12d
`and A14, ref. 2) were used as primers on the complementary
`strand and there was no final digestion step to cut between the
`
`PGDX EX. 1016
`Page 3 of 5
`
`

`

`5466
`
`Biochemistry: Sanger et al.
`primer and the newly synthesized DNA. This is the most simple
`and rapid procedure, requiring only a preliminary annealing
`of template and primer, incubation of the four separate samples
`with DNA polymerase and appropriate triphosphates, followed
`by a chase with unlabeled dATP and application to the gel for
`electrophoresis. In these experiments the inhibitors used were
`ddGTP, ddATP, ddTTP, and araCTP. The conditions used for
`the "T" samples were not entirely optimal, resulting in the
`faster-moving bands being relatively weak.
`The sequences can be read with reasonable accuracy starting
`at 88 nucleotides from the 5' end of the primer for about 80
`nucleotides (apart from some difficulty at position 3459 with
`A12d). For the next 50 nucleotides there is some uncertainty
`in the number of nucleotides in "runs" because bands are not
`actually resolved.
`With longer restriction enzyme fragments as primers it is
`necessary to split them off from the newly synthesized DNA
`chains before the electrophoresis. This is normally done by di-
`gestion with a restriction enzyme. Fig. 2 shows such an exper-
`iment in which fragment R4 was used as primer on the com-
`plementary strand of OX174 DNA. In this experiment only
`dideoxynucleoside triphosphates were used as inhibitors because
`the results with araC were much less satisfactory when a re-
`striction enzyme was used for the subsequent splitting. This may
`be due to the araC being removed by the 3'-exonuclease activity
`of the DNA polymerase during the incubation at 370 (which
`is necessary for the restriction enzyme splitting), resulting in
`a few C bands being either very faint or missing. Alternatively,
`the enzyme may be able to extend some chains beyond the araC
`at the higher temperature while being unable to do so at lower
`temperatures. araATP, which has been used only under these
`conditions, shows the same limitations as araCTP. These
`problems do not arise when ddCTP is used in this reaction.
`With one exception (positions 4330-4343, see below), a se-
`quence of 120 nucleotides, starting at a position 61 nucleotides
`from the restriction enzyme splitting site, could be read off; the
`sequence agreed with the published one. This region is believed
`to contain the origin of viral strand replication (2, 14). The
`bands beyond position 4380 indicated that there was an error
`in the provisional sequence (2), and further work (to be pub-
`lished later) has shown that the trinucleotide C-G-C should be
`inserted between positions 4380 and 4381.
`When this technique is used the products are cut with a re-
`striction enzyme as above, difficulties arise if there is a second
`restriction enzyme site close to the first one, because this will
`give rise to a separate pattern of bands that is superimposed on
`the normal one, making interpretation impossible. One way in
`which this can be avoided is by the single-site ribo-substitution
`method (N. L. Brown, unpublished). After annealing of the
`template and primer a single ribonucleotide is incorporated by
`incubation with DNA polymerase in the presence of manganese
`and the appropriate ribonucleoside triphosphate. Extension of
`the primer is then carried out with the separate inhibitors as
`above and the primer is split off at the ribonucleotide by ribo-
`nuclease or alkali. The method is particularly suitable for use
`with fragments obtained with the restriction enzyme Alu,
`which splits at the tetranucleotide sequence A-G-C-T. This
`enzyme is in fact inhibited by single-stranded DNA and cannot
`be used for the subsequent splitting of the primer from the
`newly synthesized DNA chain. The initial incorporation is
`carried out in the presence of rCTP and [a-32PldTTP. The in-
`corporation of the 32p facilitates subsequent purification on the
`Sephadex column.
`Fig. 3 shows an example of the use of this method with
`fragment A8 on the viral strand of OX174 DNA. A sequence
`
`Proc. Natl. Acad. Sci. USA 74 (1977)
`
`of about 110 nucleotides starting 33 residues from the priming
`site can be read off. In the provisional sequence (2) this region
`was regarded as very tentative. Most of it is confirmed by this
`experiment, but there is a clear revision required at positions
`3524-3530. The sequence of the viral strand should read A-
`T-C-A-A-C, replacing A-T-T-C- - -A-C given in the provisional
`sequence. There is difficulty in reading the sequence at
`3543-3550, where there is considerable variation in the distance
`between bands, suggesting the presence of a looped structure.
`Further work in which the electrophoresis was carried out at
`a higher temperature indicates that the sequence here is actu-
`ally G-C-T-C-G-C-G (viral strand); i.e., an insertion of C be-
`tween positions 3547 and 3548 in the provisional sequence.
`DISCUSSION
`The method described here has a number of advantages over
`the plus and minus methods. First, it is simpler to perform be-
`cause it requires no preliminary extension, thus avoiding one
`incubation and purification on a Sephadex column. It requires
`only the commercially available DNA polymerase I (Klenow
`fragment). The results appear to be more clear-cut with fewer
`artefact bands, and can usually be read further than with the
`plus and minus methods. Intermediate nucleotides in "runs"
`show up as bands, thus avoiding a source of error in the plus and
`minus method-estimating the number of nucleotides in a run.
`Theoretically one would expect the different bands in a run to
`be of the same strength, but this is not always the case. Fre-
`quently, the first nucleotide is the strongest, but in the case of
`ddCTP the second is the strongest (see Fig. 2). The reasons for
`these effects are not understood, but they do not usually cause
`difficulties with deducing the sequences. For the longer se-
`quences in which the separate bands in a run are not resolved,
`experience has shown that it is frequently possible to estimate
`the number of nucleotides from the strength and width of the
`band.
`The inhibitor method can also be used on a smaller scale than
`the plus and minus method because better incorporation from
`32P-labeled triphosphates is obtained. This is presumably due
`to the longer incubation period used, which allows a more
`quantitative extension of primer chains.
`In general, sequences of from 15 to about 200 nucleotides
`from the priming site can be determined with reasonable ac-
`curacy using a single primer. Frequently it is possible to read
`the gels further and, on occasions, a sequence of about 300
`nucleotides from the priming site has been determined. Oc-
`casional artefacts are observed, but these can usually be readily
`identified. It seems likely that these are usually due to con-
`taminants in the fragments. The most serious difficulties are
`due to "pile-ups" of bands, which are usually caused by the
`DNA forming base-paired loops under the conditions of the
`acrylamide gel electrophoresis. These pile-ups are seen as a
`number of bands in the same position or unusually close to one
`another on the electrophoresis. They generally occur at dif-
`ferent positions when the priming is carried in opposite direc-
`tions along the DNA over the same sequence. An example of
`this effect is seen in Fig. 2 at position 4330, where there is a
`single strong band in the G channel that in fact represents four
`G residues. They are presumably forming a stable loop by
`pairing with the four Cs at positions 4323-4326. Another ex-
`ample is in Fig. 3 at positions 3545-3550. This effect is likely
`to be found in all the rapid techniques that use gel electro-
`phoresis.
`It is felt that for an accurate determination of sequence one
`should not rely completely on single results obtained by this
`method alone but that confirmation should be obtained by some
`
`PGDX EX. 1016
`Page 4 of 5
`
`

`

`Biochemistry: Sanger et al.
`
`Proc. Nat!. Acad. Sci. USA 74 (1977)
`
`5467
`
`5.
`6.
`
`7.
`8.
`
`9.
`10.
`
`11.
`12.
`
`13.
`
`14.
`
`& Moffatt, J. G. (1969) Biochemistry 8, 4897-4904.
`Hunter, T. & Francke, B. (1975) J. Virol. 15, 759-775.
`Russell, A. F. & Moffatt, J. G. (1969) Biochemistry 8, 4889-
`4896.
`Geider, K. (1974) Eur. J. Biochem. 27,555-563.
`McCarthy, J. R., Robins, M. J., Townsend, L. B. & Robins, R. K.
`(1966) J. Am. Chem. Soc. 88, 1549-1553.
`Tener, G. M. (1961) J. Am. Chem. Soc. 83, 159-168.
`Hoard, D. E. & Ott, D. G. (1965) J. Am. Chem. Soc. 87,1785-
`1788.
`Buchi, H. & Khorana, H. G. (1972) J. Mol. Biol. 72,251-288.
`Robins, M. J., McCarthy, J. R. & Robins, R. K. (1966) Biochem-
`istry 5,224-231.
`Air, G. M.,'Sanger, F. & Coulson, A. R. (1976) J. Mol. Biol. 108,
`519-533.
`Slocombe, P. M. (1976) Ph.D. Dissertation, University of Cam-
`bridge.
`
`1.
`2.
`
`other technique or by priming on the opposite strand. This
`consideration probably applies to all other available methods
`also. The main disadvantage of the present method is the dif-
`ficulty in obtaining all the inhibitors-particularly ddGTP,
`which is not commercially available.
`We wish to thank Dr. K. Geider for a gift of ddTTP, Dr. F. E. Baralle
`for a gift of N-isobutyryl-5'-O-monomethoxytrityldeoxyguanosine,
`and Dr. M. J. Gait for useful advice on the synthetic work.
`Sanger, F. & Coulson, A. R. (1975) J. Mol. Blol. 94, 441-448.
`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) Nature 265,687-695.
`Maxam, A. M, & Gilbert, W. (1977) Proc. Nat!. Acad. Sci. USA
`74,560-564.
`Atkinson, M. R., Deutscher, M. P., Kornberg, A., Russell, A. F.
`
`3.
`
`4.
`
`PGDX EX. 1016
`Page 5 of 5
`
`