A new method for determining nucleotide sequences 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 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 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 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 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 strand separation 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.

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