Friday, 12 December 2014

Book Review: Fred Sanger's biography

Author: George G. Brownlee.
Publisher: Cambridge University Press (2014) ISBN: 9781107083349

Fred Sanger's biography has been written by his one-time PhD student, and long time collaborator, George Brownlee. Fred (I'll refer to him by his first name as I think he'd have been happy with the lower level of formality) is revealed to be a very modest individual who was happy to recognise the contributions of others around him. He refused a knighthood as he did not want to be called Sir and was quoted as saying “every one calls me Fred”, but accepted an Order of Merit in 1986. His work on the development of methods to sequence proteins and nucleic acids is described by Brownlee and is based on interviews with Fred carried out in 1992, which are also reproduced in the text. The book covers his family life, schooling and time in Cambridge as an undergraduate through to his retirement from the LMB in 1986. The book also includes commentary on the development of sequencing after completion of the Human Genome Project from fellow collaborators and Nobel laureates, Paul Berg, John Sulston and Elizabeth Blackburn.

Sanger sequencing, named after Fred, earned him his second Nobel prize, a feat achieved by only three others. It was the method used for the Human Genome Project and even though it is being superseded by next-generation sequencing methods it is difficult to understand where we would be today without it. Brownlee's introduction asks whether there was something in Fred's DNA that was behind his success, an obvious question to ask given his impact on the field of genomics but it turns out almost certainly not to be the case.

The early years and the first Nobel prize: The chapter on Fred's early years covers his home life in Rendcomb, Gloucestershire, where his father was a GP, his schooling at Bryanston boarding school, his time as an undergraduate at Cambridge after receiving a scholarship to St John's College where he met his future wife Joan Howe in the 'Scientists anti-war group', and the second world war where he undertook fire-watching duties with Joe Needham. He was raised as a Quaker and was a conscientious objector during the war. He struggled a little as an undergraduate taking an extra year before starting his Part II, but went on to complete a PhD in the Department of Biochemistry.

After his PhD Fred was offered a postion in Albert Chibnall's group where he studied the free amino groups of insulin. Over ten years from 1944 to 1954 he developed Sanger's reagent to fragment the protein into short amino-acid chains, and methods to separate these using chromatography; slowly piecing together the complete sequence of insulin and its disulfide bridge structure for which he was awarded the 1958 Nobel prize in Chemistry. This secured his reputation and his tenure in Cambridge allowing him to take risks on experiments and relieving him from teaching and grant writing duties. All at the age of just 40! Fred's success seems to have been catching: his first PhD student Rodney Porter was awarded the 1972 Nobel prize in Physiology or Medicine for his work on antibody structure. Fred admired his light-hearted attitude to research, and would comment on this  to his post-docs and students that they "should enjoy it, and if things don't work you should jolly well try something else."

There is a wonderful story from about 1958 about the first radioactive protein sequencing experiments. Fred dosed a chicken with 32P to produce radio-labelled ovalbumin, but when the chicken laid an egg the Geiger counters in the room started to scream due to the incorporation of very large amounts of phosphorus in the shell. Even in 1958 "the radiation officer was not pleased"!

On the development of Sanger sequencing and the second Nobel prize: Chapter 6 covers the development of dideoxy "Sanger" sequencing at the new Laboratory of Molecular Biology; built for Max Perutz, John Kendrew, Francis Crick and Fred. It was the arrival of a new PhD student at the LMB that would move Fred's interest from proteins to nucleic acids, specifically RNA. When George Brownlee (author) joined in 1963 he wanted to worked on the development of RNA sequencing by T1 ribonuclease digestion, it was the success of this work that led to attempts to sequence DNA. In the interviews Fred talks about the development of the dideoxy method. He admits to not making very careful notes in the early days which is astounding given the impact of this work. Prior to being interviewed by Brownlee Fred had looked back in his lab notebooks to confirm the development of the ideas for didexoy chain termination and acrylamide gel electrophoresis, which occurred in a rather roundabout way. In the early 1970s the lab had been using the ribo C method, but in 1972 or 1973 inspired by the work by Paul Englund, who had shown that T4 DNA polymerase when used with only single nucleotide would result all fragments ending in the same base due to T4 exonuclease activity, Sanger began to develop what would become the plus and minus method. Using this Fred and colleagues sequenced the 5386 bases of the PhiX genome, published in 1977 and the first genome to be completely sequenced. John Donelson in the lab took some of the samples produced for the PhiX genome, and ran them on acrylamide gels. Fred was sceptical about acrylamide, but the initial results were very promising. In the end it appears Fred was somewhat surprised that fractionation of DNA purely by size was possible, he had considered it a crazy idea. And this may explain why he did not make very careful notes on those early experiments. The work on the +/- method clearly showed that if you could get DNA fragments that differed by a single base then longer and longer reads would be possible, however the methods were laborious. Fred knew he needed a terminator nucleotide and contacted Klaus Geider who had made dideoxyTTP, results from the first tests with this first terminating nucleotide by Alan Coulson were so promising that they worked with Mike Gait in the lab to prepare the other three ddNTPs themselves. Sanger sequencing was published in 1977 in PNAS, and for this Fred was awarded his second Nobel prize in 1980.

On cloning and priming: Fred is very complementary about the work of Messing in demonstrating the cloning of dsDNA into the ssDNA bacteriophage M13. This cloning allowed the single stranded DNA to be produced for sequencing, but also allowed the use of a single universal priming strategy - the same M13fwd and M13rev many of the readers will be familiar with. This was hugely important, and often overlooked, as in 1977 the synthesis of a single short oligonucleotide took an experienced chemist about one year, compare this to ordering from Sigma or the use of long-oligos in genome synthesis today.

On genomes: The HGP was only getting off the ground in 1992 when the interviews this book is based on were conducted. Fred is quoted as saying "from the medical point of view it [the HGP] is likely to be useful" an understatement if ever there was one! In their work with the mitochondrial genome, the first in Fred's lab using M13 cloning, he and Barrell noticed that a lot of sequences contained the stop codons. They were the first to realise that the genetic code was not universal and that codon usage differed across genomes. They were also the first to report overlapping genes.

On bioinformatics: Fred was initially "a bit rude about computers"; however Roger Staden, a friend of Bart Barrell, joined the group to develop the early programs to handle sequence data. In the interview where they discuss sequencing genomes Fred argues that sequencing is a qualitative problem not a quantitative one. I wonder what he would make of the quantitative biology that is making huge strides in understanding tumour heterogeneity for instance?

On retirement: Fred Sanger retired when he got to 65. His reasons were mainly that dideoxy sequencing was a high-point to finish on and he did not want to do part-time research or move into administration. He thoroughly enjoyed his retirement and not once in the interviews appear to regret leaving research behind.

At the time of the interviews the Wellcome Trust Sanger Institute was still on the drawing board. The Sanger was built at Hinxton Hall which had already had a successful 30 years as the Tube Investment Group this was one of the first spin-outs that started the "Cambridge phenomenon", and over 34 years produced many technological innovations as well as scientific advances. Fred opened the Sanger Institute in 1993 and concluded his acceptance speech by saying "the new building could be called the 'Sanger Centre' on one condition: 'It had better be good'". The Sanger has certainly lived up to everything Fred may have hoped it would.

When writing about the award of the second Nobel one can't help but feel that George Browlee has a biased perspective, and one that is certainly different from the Nobel committee in 1980. Brownlee describes the 12 years of effort by the Sanger lab from 1965-1977 that led to the dideoxy paper, but perhaps unfairly dismisses the work of Gilbert as a single paper, and one that "most likely had benefited" from Sanger's own work. Brownlee concludes by saying "I assume the [Nobel] committee must have taken into account Gilbert's other important contributions to molecular biology" but does not elaborate (Gilbert was one of the first to propose introns, was an early proponent of the RNA world as the origin of life and is a founder of Myriad Genetics, hardly an accidental Nobel laureate). Fred himself is quoted in the text as saying "I cannot pretend that I was overjoyed by the appearance of a competitive method", but the transcript of the interview that covers this period is less controversial with Fred simply acknowledging that both contributions were recognised.

The interviews conclude with Fred being asked to look back over his career which he sums up as "being in the right place at the right time". "Maybe I've had a few ideas but I never know where they come from…" he finishes off by saying how important colleagues and students are. After all this maybe Fred's success was more a product of his Quaker upbringing; Nurture rather than Nature perhaps?

In summary: Whilst it is impossible to deny that Fred's achievements were exceptional I can't help but wonder that the development of sequencing technology would have happened even if Fred had gone into medicine as his father hoped. DNA needed to be sequenced to be understood and it surely would have been accomplished by other means had Fred not succeeded. Because of this it is a shame that Brownlee does not discuss why Sanger sequencing succeeded where Maxam and Gilbert sequencing failed? It would also like to have seen a chapter, or chapters, on the development of the next-generation of sequencing technologies with interviews of Ronaghi, Balasubramanian and Rothberg.

This short biography is written very much as the interview transcripts read, more a remembering of a dear departed friends achievements than a straight-forward analysis of the development of Sanger sequencing. A perfect Christmas present for your NGS obsessed scientist.


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  3. George Brownlee wrote that biography very well. He explained everything in depth but keeping in brief. I see this blogpost is the only source on internet to find his biography. professional bio writer

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