Frederick Sanger

Frederick Sanger was born on 13 August 1918 in Rendcomb, a small village in Gloucestershire, the second son of Frederick Sanger, a general practitioner, and his wife, Cicely Sanger née Crewdson. He was one of three children- an elder brother and a younger sister. Sanger’s father converted to Quakerism soon after his two sons were born and brought up the children as Quakers. The family were reasonably wealthy and employed a governess to teach the children. At the school he liked his teachers and particularly enjoyed scientific subjects. In college, he took courses in physics, chemistry, biochemistry and mathematics but struggled with physics and mathematics. He finally took up and graduated in biochemistry, which was a relatively new department at the time.Both his parents died from cancer during his first two years at Cambridge.

Although Sanger now happens to be an agnostic, as an undergraduate Sanger’s beliefs were strongly influenced by his Quaker upbringing. It was through his involvement with the Cambridge Scientists’ Anti-War Group that he met his future wife, Joan Howe, whom he married in 1940, and they now have three children.

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.

1958 Nobel prize in Chemistry "for his work on the structure of proteins, especially that of

insulin"

Backdrop

It was already known that different proteins had different amino acid compositions, different biological activities, and different physical properties and that genes had an important role in controlling them. But in a world of biochemistry dominated by the role of enzymes in intermediary metabolism, it was not at all clear how molecules as large as proteins could be synthesized; the idea that proteins were stochastic molecules, with a sort of "center of gravity" of structure but with appreciable microheterogeneity, was taken seriously. This is the paradigm that Fred's results shifted.

The First Sequence: Fred Sanger and Insulin

Sanger stayed in Cambridge and joined the group of Charles Chibnall, who had already done some work on the amino acid composition of bovine insulin. Chinball suggested that Sanger look at the amino groups in the protein. Insulin was one of the very few proteins that were available in a pure form.

Around 1941, Martin and Synge discovered Paper Chromatography. This was a major improvement in technique as compared to the old fractional crystallisation and precipitation to determine the peptide composition of proteins.

In 1951, Sanger determined the complete amino acid sequence of the two polypeptide chains of bovine insulin. 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.

In determining these sequences, Sanger proved that proteins have a defined chemical composition.

Sanger 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 hydrolysed 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 which Sanger called "fingerprints".

The peptide from the N-terminus could be recognised by the yellow colour imparted by the FDNB label and the identity of the labelled 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.

N-terminal

FDNB

Electrophoresis, chromatography, identification of protein at N-terminal

Partial hydrolysis

Repeat

After this success, 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.

1980, Walter Gilbert and Sanger shared half of the chemistry Nobel Prize "for

their contributions concerning the determination of base

sequences in nucleic acids".

Following the work on insulin he developed further methods for studying proteins and particularly the active centres of some enzymes.

Around 1960 he turned his attention to the nucleic acids, RNA and DNA. He developed methods for determining small sequences in RNA.

The work culminated in the development of the "dideoxy" technique for DNA sequencing around 1975.

Sanger and colleagues used this method to determine the sequence of all 5375 nucleotides of the bacteriophage _X174

This was a relatively rapid method and was used to determine the DNA sequence of the bacteriophage fx 174 of 5375 nucleotides in 1977 – this was the first complete determination of the genome of an organism, of human mitochrondrial DNA (16,338 nucleotides) and of bacteriophage l (48,500 nucleotides). The method has been much improved and automated today.

To their surprise they discovered that the coding regions of some of the genes overlapped with one another.

A DNA primer= initiates DNA synthesis

dNTPs = dATP, dCTP, dGTP, dTTP -in ALL tubes incorporated into the new DNA synthesis.

DNA polymerase!

ddNTPs = "chain terminating, or dideoxy" nucleotides in just ONE tube of 4. When incorporated, DNA synthesis on that one strand STOPS, but it continues on all other strands.

A ladder of DNA fragments of different sizes in generated (depending on the location of the chain terminating nucleotide)

Electrophoresis through thin polyacrylamide gel subjected to an electrical field. The shorter the fragment, the faster it moves through the gel. After expos use to X-ray film, presence of different sized 'bands' represents where each A, C, G, or T is located.

"The history of science is not always fair with their protagonists”...another of those great minds of science whose great achievements are not coordinated with his popular fame: Frederick Sanger, the only man to win two Nobel prizes in chemistry.

“Of the three main activities involved in scientific research, thinking, talking, and doing, I much prefer the last and am probably best at it.”

References

The First Sequence: Fred Sanger and InsulinAntony O. W. StrettonDepartment of Zoology, University of Wisconsin, Madison, Wisconsin 53706

Sequences, Sequences, and SequencesFrederick SangerRetired from Medical Research Council Laboratory of Molecular Biology, Hills Road,Cambridge CB2 2QH, England

Also, Wikipedia