Double Nobel prize-winner Fred Sanger recalls his days in the lab, attributing his success to the time he spent at the bench. Emma Davies reports
Double Nobel prize-winner Fred Sanger recalls his days in the lab, attributing his success to the time he spent at the bench. Emma Davies reports
Those who achieve great things often advise people to follow their dreams if they want to reach the same giddy heights of success. Fred Sanger, who is the only person to have twice been awarded the Nobel prize for chemistry, is no exception. In a Nobel speech in 1958, Sanger advised the student audience to ’follow those dreams as far as possible and try and do that thing that really interests you and you feel is really worthwhile’.
Throughout his working life, Sanger was guided almost solely by what really interested him. He loved ’messing about at the lab bench’ and ’got his kicks out of doing experiments’. This first led him to discover the complete structure of insulin in 1955, for which he won a Nobel prize in 1958. Instinct then took him along a different track to DNA research and to his second Nobel in 1980, awarded for so-called Sanger sequencing - a DNA decoding technique that became world famous and revolutionised genome research.
Sanger was born in 1918 in Gloucestershire, UK. His father was a general practitioner and he would have followed in his footsteps had it not been for the fact that he had witnessed the work at first hand and considered that it was ’not very pleasant and very demanding’. While at school at Bryanston, Dorset, Sanger became interested in science and decided to study natural sciences at Cambridge.
In his final year at Bryanston, knowing that he had got ’sufficient qualifications’ to get into St John’s College, Cambridge, Sanger had some time to spare. He joined his housemaster and chemistry teacher examining dyestuffs in the chemistry lab. According to his school newsletter, they applied their knowledge to catch a thief by planting some dye-coated coins in a trouser pocket.
Sanger started university a year earlier than most, armed with nothing more than a school certificate. Because his parents were fairly well off - his mother was the daughter of a wealthy cotton manufacturer - he didn’t need a scholarship. He took physics and chemistry but couldn’t really handle the physics: ’all of the other guys had done an extra year and I didn’t do terribly well’. So he changed subjects to study physiology and a subject that he had never heard of before called biochemistry.
The biochemistry department had been founded by Sir Frederick Hopkins at the turn of the century and was full of enthusiasm. Sanger was attracted by the subject’s insight into living matter and the scientific perspective that it gave to solving medical problems. He did ’pretty well’ in biochemistry and went on to specialise in the subject in his final year.
When the second world war started, Sanger chose to be a conscientious objector, a decision that was strongly influenced by the fact that his father was a very staunch Quaker. As part of the war effort, Sanger spent some time at a work camp in Devon, learning new skills such as farming, cooking, and carpentry.
While at the camp Sanger received his final’s results. ’I had got a first, which surprised me considerably!’ he recalls. This exciting news drew him back to Cambridge where he went to find out if the biochemistry department would take him on. One of the department’s researchers, Albert Neuberger, offered him a PhD working on lysine metabolism and a more practical problem concerning the nitrogen in potatoes, in a project linked to the war effort. He continued to rely on family money, which allowed him to channel all of his energy into research.
As part of the project, Sanger discovered that there is more nitrogen just beneath the potato skin than in the middle. The research was ’alright - I got a PhD’, he recalls. Although he considered the work to be rather dull, Sanger learned some invaluable lessons from Neuberger which were no doubt instrumental in making his future work such a success. He describes Neuberger as a wonderful teacher who looked after him very well.
When Sanger first started his PhD, he experienced a lack of success with his early experiments, as most students do. He recalls how ’at first I think I just did what he [Neuberger] told me. Gradually you realise that it’s up to you’. Neuberger was partly responsible for Sanger’s love of messing about at the bench, being a ’bit of a dabbler’ himself.
Insight into insulin
Sanger finished his PhD in 1943 and found himself at a bit of a loose end. Luckily, a new and interesting character, Charles Chibnall, had emerged on the scene in the biochemistry department to take over from Hopkins. He was probably the world leader in amino acid analysis, says Sanger, with a particular interest in insulin and Sanger took up the offer of a research project. Chibnall’s group had already analysed insulin’s structure and discovered that the molecule differed from most other proteins, having a high content of free α-amino groups. Chibnall suggested that Sanger should try to identify these amino acids and helped him obtain a Beit memorial fellowship for medical research to fund the work.
From the high content of free α-amino acids the researchers surmised that insulin is composed of relatively short polypeptide chains; free α-amino acid groups are only found at the end of chains. Sanger set to work trying various reagents to see if he could label the free amino groups. He came up with 1-fluoro-2,4-dinitrobenzene (FDNB), which worked rather well, reacting with the free amino groups of proteins to form dinitrophenyl (DNP) derivatives. Acid hydrolysis then gave bright yellow DNP-amino acids.
As luck would have it, Richard Synge and Archer Martin had been making great advances in using partition chromatography to separate acetyl derivatives of amino acids; they went on to win a Nobel prize in 1952 for inventing the technique. Sanger discovered that it worked ’tremendously well’ with FDNB and the researchers watched in delight as ’lovely yellow bands’ came down the column.
Sanger applied the method to insulin and discovered three yellow DNP-derivatives: ε-DNP-lysine, DNP-phenylalanine and DNP-glycine. The ε-DNP-lysine turned out to be a red herring and the team began to think that insulin was composed of four polypeptide chains, two with phenylalanine end groups and two with glycine end groups. Oxidation or varying levels of acid hydrolysis on the DNP derivatives indicated that insulin in fact comprises only two chain types.
By using a range of basic analytical techniques including ion-exchange chromatography and 2D paper chromatography, Sanger managed to elucidate the complete structure of insulin, which was published in 1955. In 1958, he was awarded the Nobel prize for chemistry for the work. Only in his dreams had he thought that he might get the prize, although he admits that ’rumours had been going around everywhere’.
In his speech at the Nobel banquet in Sweden in December 1958, Sanger voiced his hopes that the prize would encourage other protein chemists. His words echoed the lessons learnt early on in his PhD and must have been heartening for many disillusioned researchers who may have thought that a Nobel prize winner would not experience many setbacks. ’For to the scientific worker, encouragement is much appreciated. So often if one takes stock at the end of a day or a week or a month and asks oneself what have I actually accomplished during this period, the answer is often "nothing’’ or "very little’’ and one is apt to be discouraged and wonder if it is really worth all that effort that one devotes to some small detail of science that may in fact never materialise,’ said Sanger.
In his Nobel autobiography, Sanger acknowledged that the prize had given him ’renewed confidence and enthusiasm’. It also allowed him to ’obtain better research facilities and, even more importantly, to attract excellent colleagues’.
In 1962 Sanger moved to the new Medical Research Council’s Laboratory of Molecular Biology (LMB) in Cambridge. Here, a collection of talented scientists had amassed, including Francis Crick and Max Perutz. Sanger knew and respected these scientists but probably didn’t mix with them much because he gives the impression that he rarely left the confines of the lab: ’my interests were always at the lab bench doing experiments’.
My interests were always at the lab bench doing experiments
Sanger continued to work on proteins but didn’t get very far and entered what he describes as a fairly unproductive period. He thinks that he may not have got support for the project if he hadn’t been awarded a Nobel prize.
He tried to develop methods for looking at the active centres of enzymes and made some progress using paper chromatography and various other techniques. Before long the research atmosphere at the LMB had enticed him to start work on nucleic acids. At the time it seemed a fairly dramatic move, but as Sanger points out ’the concern with the basic problem of sequencing remained the same’.
Sanger initially spent some time ’fiddling around’ with transfer ribonucleotides. His research group didn’t make much progress and it soon became evident that DNA was the thing to work on because, quite simply, ’that is what we are really’.
’The trouble with DNA is the terrific size of it,’ says Sanger. With fellow researchers George Brownlee and Bart Barrell, Sanger developed a small-scale method for fractionating radioactive-labelled oligonucleotides. This used enzymes to break down the RNA into smaller products which could then be separated to give information on base sequences. Sanger and his team could then determine the base sequences and reconstruct entire sequences using the overlaps.
The technique proved slow and tedious, particularly with larger DNA molecules and it soon became apparent that they needed a new analytical method.
The breakthrough came when Sanger and his colleagues got the so-called dideoxy method working. Sanger received his second Nobel prize for this in 1980 - he shared the prize with Paul Berg and Walter Gilbert - and it is the work of which he is most proud. Commonly called Sanger sequencing, the technique allows large numbers of bases to be read and works by inserting chain-terminating nucleotide analogues into DNA during synthesis
Sanger’s team used the new technique to complete the entire genome of a bacteriophage called φX174, a tiny virus that infects bacteria. In 1981, they sequenced the first human genome, the DNA of mitochondria - energy factories in our cells which have their own genome of about 16 000 base pairs. Bacteriophage λ came next, a virus with a genome of about 48 000 base pairs, which was sequenced in 1982. To analyse this, Sanger and his team developed the shotgun method, which involves randomly sampling and determining 500-700 bases at a time before assembling them to reconstruct the sampled sequence.
Brownlee, who now works at the Sanger Institute, recalls the excitement: 'Because of Fred we always had the best technology in the world. Everybody came to you; it was an amazing feeling.’
Sanger’s work paved the way to highly automated genome sequencing, using different coloured fluorescent dyes and automatic data systems. The method is now used to attack genomes containing as many as three billion base pairs.
Sanger retired in 1983 at the age of 65, turning his back on the science that had so fascinated him to focus instead on gardening and boating. Looking back, Sanger thinks that he retired at the right time, acknowledging that ’what was needed to take the DNA sequencing technique to the next level was mass production’. This didn’t interest him because he wasn’t keen on the business side of things and was ’more of a technician than an entrepreneur’. He gives the distinct impression that he would not have been so keen on sequencing research today, given that much of it requires sitting in front of a computer screen.
Genomic research advanced so rapidly after Sanger retired that he now claims that he would struggle to read today’s journal articles in the field, mainly because the scientific language has changed so much. He may be more interested in the field than he lets on: a note about him on the Sanger Institute’s website reports that he makes ’occasional forays to the institute that bears his name to make sure all is as it should be’.
Sanger attributes his success to his time spent in the lab. ’All through my career I was at the bench. I always spent most of the time messing about with test tubes and smidgenisers.’ (LMB researchers affectionately named the piece of equipment used to analyse chromatography spots a smidgeniser.)
Sanger can still offer advice to today’s young scientists. ’You have got to get in the habit of thinking about what the next experiment is going to be and you have got to think of different approaches to problems. It comes if you’re working at the bench. You get a more practical sort of attitude - you know what is likely to work and what will not.’ Brownlee reiterates this: ’He [Sanger] once said that most experiments didn’t work but he always perservered and of course when they did work it was revolutionary’.
Working at the bench, you get a more practical attitude - you know what is likely to work and what will not
Sanger is sometimes labelled the father of modern molecular biology. He laughingly rejects this as being a bit extreme but then concedes that DNA sequencing has opened up an awful lot of research. All that time at the bench certainly paid off.
To synthesise DNA requires a supply of its four nucleotides, the enzyme DNA polymerase, and a primer, a short DNA sequence annealed to the template which initiates the new DNA strand. The nucleotides that add to the growing DNA strand are complementary to those in the template strand.
In Sanger sequencing, nucleotide analogues called dideoxynucleoside triphosphates incorporate into the growing chain. These are the same as deoxynucleoside triphosphates but lack the 3’-hydroxyl group and so act as chain terminators.
The technique uses four reactions, each of which contains the template, primer and DNA polymerase, four deoxynucleoside triphosphates, and a small amount of dideoxy chain terminator specific to A, C, G or T. One of the deoxynucleoside triphosphates is labelled with the radioactive 32P.
Most of the chains incorporate the normal nucleotides and continue to expand, but a small proportion add the chain-terminating dideoxy compounds. The resulting fragments can then be size-separated using gel electrophoresis, which separates DNA molecules according to size even if they differ in length by only a single nucleotide. All of the chains in the T reaction mixture, for example, will end at T and so the relative position of the Ts will define the chain sizes and the relative positions on the gel.