Frederick Sanger was born on 13 August 1918 in Rendcomb, a small villagein 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 youngersister. Sanger’s father converted to Quakerism soon after his two sons were born andbrought up the children as Quakers. The family were reasonably wealthy and employed agoverness to teach the children. At the school he liked his teachers and particularly enjoyedscientific subjects. In college, he took courses in physics, chemistry, biochemistry andmathematics but struggled with physics and mathematics. He finally took up and graduatedin 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 beliefswere strongly influenced by his Quaker upbringing. It was through his involvement with theCambridge Scientists’ Anti-War Group that he met his future wife, Joan Howe, whom hemarried 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 toinvestigate whether edible protein could be obtained from grass. After little more than amonth Pirie left the department and Albert Neuberger became his adviser. Sanger changedhis research project to study the metabolism of lysine and a more practical problemconcerning 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 Freds results shifted.
The First Sequence: Fred Sanger and InsulinSanger stayed in Cambridge and joined the group of Charles Chibnall, who hadalready done some work on the amino acid composition of bovine insulin.Chinball suggested that Sanger look at the amino groups in the protein. Insulinwas one of the very few proteins that were available in a pure form.Around 1941, Martin and Synge discovered Paper Chromatography. This was amajor improvement in technique as compared to the old fractionalcrystallisation and precipitation to determine the peptide composition ofproteins.In 1951, Sanger determined the complete amino acid sequence of the twopolypeptide chains of bovine insulin.Sangers principal conclusion was that the two polypeptide chains of the proteininsulin had precise amino acid sequences and, by extension, that every proteinhad a unique sequence.In determining these sequences, Sanger proved that proteins have a definedchemical composition.
Sanger used the "Sanger Reagent", fluorodinitrobenzene (FDNB), to react with the exposedamino groups in the protein and in particular with the N-terminal amino group at one end ofthe polypeptide chain.He then partially hydrolysed the insulin into short peptides (either with hydrochloric acid orusing an enzyme such as trypsin). The mixture of peptides was fractionated in twodimensions 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 ofinsulin, detected with ninhydrin, moved to different positions on the paper, creating adistinct pattern which Sanger called "fingerprints".The peptide from the N-terminus could be recognised by the yellow colour imparted by theFDNB label and the identity of the labelled amino acid at the end of the peptide determinedby complete acid hydrolysis and discovering which dinitrophenyl-amino acid was there. Byrepeating this type of procedure Sanger was able to determine the sequences of the manypeptides generated using different methods for the initial partial hydrolysis. These couldthen be assembled into the longer sequences to deduce the complete structure of insulin.
N-terminal FDNBElectrophoresis, chromatography, identification of protein at N-terminal Partial hydrolysis Repeat
After this success, he started looking at the possibility of sequencingRNA molecules and began developing methods for separatingribonucleotide fragments generated with specific nucleases. One ofthe problems was to obtain a pure piece of RNA to sequence. In thecourse of this he discovered in 1964, with Kjeld Marcker, theformylmethionine tRNA which initiates protein synthesis in bacteria.He was beaten in the race to be the first to sequence a tRNAmolecule by a group led by Robert Holley from Cornell Universitywho published the sequence of the 77 ribonucleotides of alaninetRNA from Saccharomyces cerevisiae in 1965. By 1967 Sangers group had determined the nucleotide sequence ofthe 5S ribosomal RNA from Escherichia coli, a small RNA of 120nucleotides.
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 forstudying 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" techniquefor DNA sequencing around 1975.Sanger and colleagues used this method to determine the sequence ofall 5375 nucleotides of the bacteriophage _X174This was a relatively rapid method and was used to determine the DNAsequence of the bacteriophage fx 174 of 5375 nucleotides in 1977 – thiswas the first complete determination of the genome of an organism, ofhuman mitochrondrial DNA (16,338 nucleotides) and ofbacteriophage l (48,500 nucleotides). The method has been muchimproved and automated today.To their surprise they discovered that the coding regions of some of thegenes overlapped with one another.
A DNA primer= initiates DNA synthesisdNTPs = 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. Whenincorporated, 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 thechain terminating nucleotide)Electrophoresis through thin polyacrylamide gel subjected to an electrical field. The shorterthe fragment, the faster it moves through the gel. After expos use to X-ray film, presence ofdifferent sized bands represents where each A, C, G, or T is located.
"The history of science is not always fair withtheir protagonists”...another of those great minds of sciencewhose great achievements are not coordinatedwith his popular fame: Frederick Sanger, theonly man to win two Nobel prizes inchemistry.
“Of the three main activitiesinvolved in scientific research,thinking, talking, and doing, Imuch prefer the last and amprobably best at it.”
ReferencesThe First Sequence: Fred Sanger and InsulinAntony O. W. StrettonDepartment of Zoology, University ofWisconsin, Madison, Wisconsin 53706Sequences, Sequences, and SequencesFrederick SangerRetired from Medical Research CouncilLaboratory of Molecular Biology, HillsRoad,Cambridge CB2 2QH, EnglandAlso, Wikipedia