1. Frederick Sanger

2. 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.

3. 1958 Nobel prize in Chemistry "for his work on
the structure of proteins, especially that of
insulin"

4. 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.

5. 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.

6. 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.

7. N-terminal
FDNB
Electrophoresis, chromatography, identification of protein at N-terminal
Partial hydrolysis
Repeat

8. 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.

9. 1980, Walter Gilbert and Sanger shared half
of the chemistry Nobel Prize "for their
contributions concerning the determination of
base sequences in nucleic acids".

10. 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.

11. 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.

13. "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.

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

15. References
The First Sequence: Fred Sanger and Insulin
Antony O. W. Stretton
Department of Zoology, University of
Wisconsin, Madison, Wisconsin 53706
Sequences, Sequences, and Sequences
Frederick Sanger
Retired from Medical Research Council
Laboratory of Molecular Biology, Hills
Road,
Cambridge CB2 2QH, England
Also, Wikipedia 