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 ABSTRACT A new method for determining nucleotide se- a stereoisomer of ribose in which the 3'-hydroxyl group is ori- quences in DNA is described. It is similar to the "plus and ented in trans position with respect to the 2'-hydroxyl group. minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94,441-4481 but makes use of the 2',3'-dideoxy and arabinonu- The arabinosyl (ara) nucleotides act as chain terminating in- cleoside analogues ofthe normal deoxynucleoside triphosphates, hibitors of Escherichia coli DNA polymerase I in a manner which act as specific chain-terminating inhibitors of DNA comparable to ddT (4), although synthesized chains ending in polymerase. The technique has been applied to the DNA of 3' araC can be further extended by some mammalian DNA bacteriophage 4bX174 and is more rapid and more accurate than polymerases (5). In order to obtain a suitable pattern of bands either the plus or the minus method. from which an extensive sequence can be read it is necessary to have a ratio of terminating triphosphate to normal triphos- The "plus and minus" method (1) is a relatively rapid and phate such that only partial incorporation of the terminator simple technique that has made possible the determination of occurs. For the dideoxy derivatives this ratio is about 100, and the sequence of the genome of bacteriophage 4X174 (2). It for the arabinosyl derivatives about 5000. 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 METHODS available techniques, neither the "plus" nor the "minus" Preparation of the Triphosphate Analogues. The prepa- method is completely accurate, and in order to establish a se- ration of ddTTP has been described (6, 7), and the material is quence both must be used together, and sometimes confirma- now commercially available. ddA has been prepared by tory data are necessary. W. M. Barnes (J. Mol. Biol., in press) McCarthy et al. (8). We essentially followed their procedure has recently developed a third method, involving ribo-substi- and used the methods of Tener (9) and of Hoard and Ott (10) tution, which has certain advantages over the plus and minus to convert it to the triphosphate, which was then purified on method, but this has not yet been extensively exploited. DEAE-Sephadex, using a 0.1-1.0 M gradient of triethylamine Another rapid and simple method that depends on specific carbonate at pH 8.4. The preparation of ddGTP and ddCTP chemical degradation of the DNA has recently been described has not been described previously; however we applied the by Maxam and Gilbert (3), and this has also been used exten- same method as that used for ddATP and obtained solutions sively for DNA sequencing. It has the advantage over the plus having the requisite terminating activities. The yields were very and minus method that it can be applied to double-stranded low and this can hardly be regarded as adequate chemical DNA, but it requires a strand separation or equivalent frac- characterization. However, there can be little doubt that the tionation of each restriction enzyme fragment studied, which activity was due to the dideoxy derivatives. makes it somewhat more laborious. The starting material for the ddGTP was N-isobutyryl-5'- This paper describes a further method using DNA poly- O-monomethoxytrityldeoxyguanosine prepared by F. E. merase, which makes use of inhibitors that terminate the newly Baralle (11). After tosylation of the 3'-OH group (12) the synthesized chains at specific residues. compound was converted to the 2',3'-didehydro derivative with Principle of the Method. Atkinson et al. (4) showed that the sodium methoxide (8). The isobutyryl group was partly re- inhibitory activity of 2',3'-dideoxythymidine triphosphate moved during this treatment and removal was completed by (ddTTP) on DNA polymerase I depends on its being incorpo- incubation in NH3 (specific gravity 0.88) overnight at 45°. The rated into the growing oligonucleotide chain in the place of didehydro derivative was reduced to the dideoxy derivative (8) thymidylic acid (dT). Because the ddT contains no 3'-hydroxyl and converted to the triphosphate as for the ddATP. The mo- group, the chain cannot be extended further, so that termination nophosphate was purified by fractionation on a DEAE-Se- occurs specifically at positions where dT should be incorporated. phadex column using a triethylamine carbonate gradient If a primer and template are incubated with DNA polymerase (0.025-0.3 M) but the triphosphate was not purified. in the presence of a mixture of ddTTP and dTTP, as well as the ddCTP was prepared from N-anisoyl-5'-O-monomethoxy- other three deoxyribonucleoside triphosphates (one of which trityldeoxycytidine (Collaborative Research Inc., Waltham, is labeled with 32p), a mixture of fragments all having the same MA) by the above method but the final purification on 5' and with ddT residues at the 3' ends is obtained. When this DEAE-Sephadex was omitted because the yield was very low mixture is fractionated by electrophoresis on denaturing and the solution contained the required activity. The solution acrylamide gels the pattern of bands shows the distribution of was used directly in the experiments described in this paper. dTs in the newly synthesized DNA. By using analogous ter- An attempt was made to prepare the triphosphate of the minators for the other nucleotides in separate incubations and intermediate didehydrodideoxycytidine because Atkinson et 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 Abbreviations: The symbols C, T, A, and G are used for the deoxyri- other rapid techniques mentioned above. bonucleotides in DNA sequences; the prefix dd is used for the 2',3'- Two types of terminating triphosphates have been used-the dideoxy derivatives (e.g., ddATP is 2',3'-dideoxyadenosine 5'-tri- dideoxy derivatives and the arabinonucleosides. Arabinose is phosphate); the prefix ara is used for the arabinose analogues. 5463 Downloaded by guest on September 30, 2021 5464 Biochemistry:' Sanger et al. Proc. Natl. Acad. Sci. USA 74 (1977) A14 (-) A 12d(- G A T C G A T C :.. S: _ | < .. _ .. C,-t;ri. | 0*.. T aA 4040 I I AxA GAC~ ~ 40320 /t -'~ (-AG-(" QiT t e _ P-t..A'AAAIT 35,10 4Ozc20C1 1-Ta G-A- 2:e. IT AT A 3500 401A0A1(A OFO A UAs4A33)40 44 -1A;AT c At A ( $48( jij~~~C.'G 4000 T(,1 Q ( 1 04-A-A-- - 3999)(;001(oT I-1A 1 A- - ; jA 7T-3- by C 34980C0 39601AT -C A 3ox4810 ~~~~~~~( AC 34 7 T A 39T0 -t it1-to3430 A A A 3460 3950Q( ACs. iAA o A A _ Ad GAT 34150 3940 GrT AC AC 34 300 -CA 3440 3930 T A 4.m. C A A 3420 A AA 3 FIG. 1. Autoradiograph of the acrylamide gel from the sequence determination using restriction fragments A12d and A14 as primers on the complementary strand of IX174 DNA. The inhibitors used were (left to right) ddGTP, ddATP,~ACTGAddTTP, 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 ddG: 0.1 mM dCTP, 0.1 mM dTTP, 0.005 mM dGTP, 0.5 as a terminator. However, we were unsuccessful in this. These mM ddGTP compounds seem much less stable than the dideoxy deriva- ddC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP, tives. approximately 0.25 mM ddCTP araATP and araCTP were obtained from P-L Biochemicals (The concentration of the ddCTP was uncertain because there Inc., Milwaukee, WI. was insufficient yield to determine it, but the required dilution Sequencing Procedure. Restriction enzyme fragments were of the solution was determined experimentally.) obtained from OX174 replicative form and separated by elec- araC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP, 12.5 trophoresis on acrylamide gels. The material obtained from 5 mM araCTP ,ug of OX174 replicative form in 5 ,Al of H20 was mixed with Incubation was at room temperature for 15 min. Then 1 Ml 1 Al of viral or complementary strand 4X174 DNA (0.6 jg) and of 0.5 mM dATP was added and incubation was continued for 1 Mi of H X 10 buffer (13) and sealed in a capillary tube, heated a further 15 min. If this step (chase) was omitted some termi- to 1000 for 3 min, and then incubated at 670 for 30 min. The nation at A residues occurred in all samples due to the low solution was diluted to 20 Al with H buffer and 2 Al samples concentration of the [a-32PldATP. With small primers, where were taken for each incubation and mixed with 2 Ml of the ap- it was unnecessary to carry out a subsequent splitting (as in the propriate "mix" and 1 il of DNA polymerase (according to experiment shown in Fig.