Frederick Sanger (1918-2013) Genetics Pioneer, Two-Time Winner of Nobel Prize

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Nobel prize-winning scientist Frederick Sanger pictured at home in 1993 at age 75.
Photograph: David Levenson/Getty Images

<<I heard a story that when Fred Sanger was working on dideoxy sequencing he didn’t publish a paper for 7 years. He got flak from his head of department for his lack of productivity only to respond by publishing his method, changing molecular biology forever and winning a second Nobel Prize.>> Comment from a Guardian reader, November 2013

 

Colin Blakemore, professor of neuroscience and philosophy at the School of Advanced Study in London and former chief executive of the Medical Research Council, said: “The death of a great person usually provokes hyperbole, but it is impossible to exaggerate the impact of Fred Sanger’s work on modern biomedical science. His invention of the two critical technical advances – for sequencing proteins and nucleic acids – opened up the fields of molecular biology, genetics and genomics. He remains the only person to have won two Nobel prizes in chemistry – recognizing his unique contribution to the modern world.“

 

Sanger was awarded a share of the Nobel prize for chemistry in 1980 for his work on sequencing DNA. It was his second Nobel prize, having also won the chemistry award in 1958 for his pioneering work on the structure of the protein insulin. He is one of only four people to have won two Nobel prizes – the highest honors in science – and the only person to have won two Nobel prizes in chemistry.

 

Compared with his contemporaries, the discoverers of the structure of DNA, James Watson and Francis Crick, Sanger was a relatively unknown figure outside science. He never courted fame (describing himself as “a chap who messed about in his lab“) and retired at the age of 65 to devote time to his garden. He even rejected a knighthood because, he told a journalist in 2000, he did not care to be called Sir. He was awarded the Order of Merit by the Queen in 1986.

 

Reading the DNA letters that make up genes of living organisms is done routinely in modern laboratories, and understanding how particular sequences influence a person’s susceptibility to diseases such as cancer and heart disease is a major focus of medical research. Though Watson and Crick had worked out the structure of DNA’s double helix in the early 1950s, and revealed that it held a linear code of base pairs (C, G, T and A), it took Sanger and his team at Cambridge to work out a way to read the DNA sequence. In the 1960s and 70s, Sanger developed techniques to clone the DNA of the genes under investigation and then add chemicals to break it into short pieces. Sanger’s group were the first to produce a whole genome sequence – 5,000 letters long of the virus phiX174 – and they also sequenced the first bit of human genetic material, the 16,000-letter sequence of DNA in a mitochondrion, the “batteries“ inside biological cells.

 

In 1962, he moved to the new Medical Research Council Laboratory of Molecular Biology along with leading scientists including Nobel laureates Max Perutz and Francis Crick and began working on the problem of sequencing DNA. His technique – “dideoxy“ or “Sanger“ sequencing – is still in use today to read DNA code, including the 3bn base pairs of the first ever complete human genome sequence published in 2003.

 

Sanger was the fourth person to have been awarded two Nobel Prizes, either individually or in tandem with others.

 

When Sanger was around five years old the family moved to the small village of Tanworth-in-Arden in Warwickshire. The family were reasonably wealthy and employed a governess to teach the children. In 1927, at the age of nine, he was sent to the Downs School, a residential preparatory school run by Quakers near Malvern. His brother Theo was a year ahead of him at the same school. In 1932, at the age of 14, he was sent to the recently established Bryanston School in Dorset. This used the Dalton system and had a more liberal regime which Sanger much preferred. At the school he liked his teachers and particularly enjoyed scientific subjects.

 

He achieved good results in the School Certificate examinations and in 1936 moved as an undergraduate to St John’s College, Cambridge to study natural sciences. His father had attended the same college. For Part I of his Tripos (The University of Cambridge, divides the different kinds of honors bachelor’s degree by Tripos, plural Triposes) he took courses in physics, chemistry, biochemistry and mathematics but struggled with physics and mathematics. Many of the other students had studied more mathematics at school. In his second year he replaced physics with physiology. He took three years to obtain his Part I. For his Part II he studied biochemistry. It was a relatively new department founded by Gowland Hopkins with enthusiastic lecturers who included Malcolm Dixon, Joseph Needham and Ernest Baldwin.

 

Sanger began studying for a PhD in October 1940 under N.W. “Bill“ Pirie. His project was to investigate whether edible protein could be obtained from grass. After little more than a month Pirie left the department and Albert Neuberger became his adviser. Sanger changed his research project to study the metabolism of lysine and a more practical problem concerning the nitrogen of potatoes. His thesis had the title, “The metabolism of the amino acid lysine in the animal body“. He was examined by Charles Harington and Albert Charles Chibnall and awarded his doctorate in 1943.

 

Neuberger moved to the National Institute for Medical Research in London, but Sanger stayed in Cambridge and in 1943 joined the group of Charles Chibnall, a protein chemist who had recently taken up the chair in the Department of Biochemistry. Chibnall had already done some work on the amino acid composition of bovine insulin and suggested that Sanger look at the amino groups in the protein. Insulin could be purchased from Boots (Boots UK Limited, formerly Boots the Chemist is a pharmacy chain in the United Kingdom) and was one of the very few proteins that were available in a pure form. Up to this time Sanger had been funding himself. In Chibnall’s group he was initially supported by the Medical Research Council and then from 1944 until 1951 by a Beit Memorial Fellowship for Medical Research.

 

Sanger’s first triumph was to determine the complete amino acid sequence of the two polypeptide chains of bovine insulin, A and B, in 1952 and 1951, respectively. Prior to this it was widely assumed that proteins were somewhat amorphous. In determining these sequences, Sanger proved that proteins have a defined chemical composition. For this purpose he used the “Sanger Reagent“, fluorodinitrobenzene (FDNB), to react with the exposed amino groups in the protein and in particular with the N-terminal amino group at one end of the polypeptide chain. He then partially hydrolyzed the insulin into short peptides, either with hydrochloric acid or using an enzyme such as trypsin. The mixture of peptides was fractionated in two dimensions on a sheet of filter paper, first by electrophoresis in one dimension and then, perpendicular to that, by chromatography in the other. The different peptide fragments of insulin, detected with ninhydrin, moved to different positions on the paper, creating a distinct pattern that Sanger called “fingerprints“. The peptide from the N-terminus could be recognized by the yellow color imparted by the FDNB label and the identity of the labeled amino acid at the end of the peptide determined by complete acid hydrolysis and discovering which dinitrophenyl-amino acid was there. By repeating this type of procedure Sanger was able to determine the sequences of the many peptides generated using different methods for the initial partial hydrolysis. These could then be assembled into the longer sequences to deduce the complete structure of insulin. Finally, because the A and B chains are physiologically inactive without the three linking disulfide bonds (two interchain, one intrachain on A), Sanger and coworkers determined their assignments in 1955. Sanger’s principal conclusion was that the two polypeptide chains of the protein insulin had precise amino acid sequences and, by extension, that every protein had a unique sequence. It was this achievement that earned him his first Nobel prize in Chemistry in 1958. This discovery was crucial for the later sequence hypothesis of Crick for developing ideas of how DNA codes for proteins.

 

From 1951 Sanger was a member of the external staff of the Medical Research Council and when they opened the Laboratory of Molecular Biology in 1962, he moved from his laboratories in the Biochemistry Department of the university to the top floor of the new building. He became head of the Protein Chemistry division. Soon after his move he started looking at the possibility of sequencing RNA molecules and began developing methods for separating ribonucleotide fragments generated with specific nucleases. One of the problems was to obtain a pure piece of RNA to sequence. In the course of this he discovered in 1964, with Kjeld Marcker, the formylmethionine tRNA which initiates protein synthesis in bacteria. He was beaten in the race to be the first to sequence a tRNA molecule by a group led by Robert Holley from Cornell University, who published the sequence of the 77 ribonucleotides of alanine tRNA from Saccharomyces cerevisiae in 1965. By 1967 Sanger’s group had determined the nucleotide sequence of the 5S ribosomal RNA from Escherichia coli, a small RNA of 120 nucleotides.

 

He then turned to sequencing DNA, which would require an entirely different approach. He looked at different ways of using DNA polymerase I from E. coli to copy single stranded DNA. In 1975 together with Alan Coulson he published a sequencing procedure using DNA polymerase with radio labeled nucleotides that he called the “Plus and Minus“ technique. This involved two closely related methods that generated short oligonucleotides with defined 3′ termini. These could be fractionated by electrophoresis on a polyacrylamide gel and visualized using autoradiography. The procedure could sequence up to 80 nucleotides in one go and was a big improvement on what had gone before, but was still very laborious. Nevertheless, his group were able to sequence most of the 5,386 nucleotides of the single-stranded bacteriophage phagX174. This was the first fully sequenced DNA-based genome. To their surprise they discovered that the coding regions of some of the genes overlapped with one another.

 

In 1977 Sanger and colleagues introduced the “dideoxy“ chain-termination method for sequencing DNA molecules, also known as the “Sanger method“. This was a major breakthrough and allowed long stretches of DNA to be rapidly and accurately sequenced. It earned him his second Nobel prize in Chemistry in 1980, which he shared with Walter Gilbert and Paul Berg. The new method was used by Sanger and colleagues to sequence human mitochondrial DNA (16,569 base pairs) and bacteriophage ? (48,502 base pairs). The dideoxy method was eventually used to sequence the entire human genome.

 

As of 2013, he is the only person to have been awarded the Nobel Prize in Chemistry twice, and one of only four two-time Nobel laureates: The other three were Marie Curie (Physics, 1903 and Chemistry, 1911), Linus Pauling (Chemistry, 1954 and Peace, 1962) and John Bardeen (twice Physics, 1956 and 1972).

 

Sanger married Margaret Joan Howe in 1940. They had three children ? Robin, born in 1943, Peter born in 1946 and Sally Joan born in 1960. He said that his wife had “contributed more to his work than anyone else by providing a peaceful and happy home.

 

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The Sanger Institute

 

Sanger retired in 1983 to his home, “Far Leys“, in Swaffham Bulbeck outside Cambridge and next to Sanger Wood. In 1992, the Wellcome Trust and the Medical Research Council founded the Sanger Centre (now the Sanger Institute), named after him. The Institute is located on the Wellcome Trust Genome Campus near Hinxton, only a few miles from Sanger’s home. He agreed to having the Centre named after him when asked by John Sulston, the founding director, but warned, “It had better be good.“ It was opened by Sanger in person on 4 October 1993, with a staff of fewer than 50 people, and went on to take a leading role in the sequencing of the human genome. The Institute now has over 900 people and is one of the world’s largest genomic research centers.

 

He declined the offer of a knighthood, as he did not wish to be addressed as “Sir“. He is quoted as saying, “A knighthood makes you different, doesn’t it, and I don’t want to be different.“ In 1986, he accepted the award of an Order of Merit, which can have only 24 living members.

 

In 2007 the British Biochemical Society was given a grant by the Wellcome Trust to catalogue and preserve the 35 laboratory notebooks in which Sanger recorded his remarkable research from 1944 to 1983. In reporting this matter, Science noted that Sanger, “the most self-effacing person you could hope to meet“, was now spending his time gardening at his Cambridgeshire home.

 

In 2008, and with his express consent, NHS Gloucestershire Primary Care Trust named their newly built HQ building Sanger House in recognition of his pre-eminent work and as a citizen of the county.

 

Sanger died on 19 November 2013. As The Times noted in his obituary, he had described himself as “just a chap who messed about in a lab“.

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Keystone/Hulton Archive, via Getty Images. Dr. Sanger in 1958 at his home in Cambridge, England.

 

Sources: The New York Times, by Denise Gellene; Daniel E. Slotnik contributed reporting, Wikipedia, The Guardian, The Sanger Institute

 

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