Determination of nucleotide sequences in DNA.

In spite of the important role played by DNA sequences in living matter, it is only relatively recently that general methods for their determination have been developed. This is mainly because of the very large size of DNA molecules, the smallest being those of the simple bacteriophages such as qXl74 (which contains about 5,000 nucleotides). It was therefore difficult to develop methods with such complicated systems. There are however some relatively small RNA molecules notably the transfer RNAs of about 75 nucleotides, and these were used for the early studies on nucleic acid sequences (1). Following my work on amino acid sequences in proteins (2) I turned my attention to RNA and, with G.G. Brownlee and B.G. Barrell, developed a relatively rapid small-scale method for the fractionation of P-labelled oligonucleotides (3). This became the basis for most subsequent studies of RNA sequences. The general approach used in these studies, and in those on proteins, depended on the principle of partial degradation. The large molecules were broken down, usually by suitable enzymes, to give smaller products which were then separated from each other and their sequence determined. When sufficient results had been obtained they were fitted together by a process of deduction to give the complete sequence. This approach was necessarily rather slow and tedious, often involving successive digestions and fractionations, and it was not easy to apply it to the larger DNA molecules. When we first studied DNA some significant sequences of about 50 nucleotides in length were obtained with this method (4,5), but it seemed that to be able to sequence genetic material a new approach was desirable and we turned our attention to the use of copying procedures.