Metodo Sanger

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 fordetermining nucleotide seABSTRACT 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 arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA ofbacteriophage 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 methodis 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 sequence both must be used together, and sometimes confirmatory data are necessary. W. M. Barnes (J. Mol. Biol., in press) has recently developed a third method, involving ribo-substitution, which has certain advantages over the plusand 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 extensively 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 strandseparation or equivalent fractionation of each restriction enzyme fragment studied, which makes it somewhat more laborious. This paper describes a further method using DNA polymerase, 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 incorporated 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 ddTTPand 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 terminatorsfor 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
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a stereoisomer of ribose in which the 3'-hydroxyl groupis oriented in trans position with respect to the 2'-hydroxyl group. The arabinosyl (ara) nucleotides act as chain terminating inhibitors 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...