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Before coming to the new LMB building, Fred Sanger had not had much interest in nucleic acids, “however, with people like Francis Crick around it was difficult to ignore nucleic acids or to fail to realize the importance of sequencing them”. There was also a strong influence and help from John Smith, who was the Laboratory expert in nucleic acid chemistry. There were compositional differences from sequencing proteins - since there are only four monomers, larger degradation products are needed to establish significant overlaps, but with only four components the final analyses are easier. He first turned his attention to RNA and, with G. G. Brownlee and B. G. Barrell, developed a relatively rapid small-scale method for the fractionation of 32p-labelled oligonucleotides. This involved breaking down the RNA into small pieces, separating them, sequencing them and fitting them together from overlaps. It was not easy to apply to the larger DNA molecules. |
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Principle of the Chain-terminating (dideoxy Method for Sequencing DNA. |
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After feeling the way with other slower and less accurate techniques, the “dideoxy” method was developed. In this, a section of the DNA is copied but using deoxynucleoside triphosphates which can be labelled with 32p, and a small amount of the dideoxy triphosphate of one of the bases - say thymidine (ddTTP). Most of the chains will incorporate the normal substrate T and be extended further, but a small proportion will add ddTTP and be unable to grow. Similar incubations are made with the dideoxy triphosphates of the other bases. The four mixtures are fractionated on acrylamide gel under denaturing conditions which separates the chains according to size. All the chains in the T mixture end in T and so their relative positions in the chain will be indicated after fractionation. Thus the actual sequence can be read off from an autoradiograph of a gel of the four mixtures in adjacent lanes. Fred Sanger was elected a Fellow of the Royal Society in 1954, awarded a CBE in 1963, became a Companion of Honour in 1981, and was appointed to the Order of Merit in 1986. |
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