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Before the 1950s, biochemists knew proteins were chains of amino acids but could not say in what order. Before the 1970s, nobody could read the order of bases in a gene quickly. Frederick Sanger solved both problems, twenty years apart, with the same basic strategy. This post explains how, with a worked example of reading a sequencing gel.
Who was Frederick Sanger?
The Nobel Prize facts page records that Sanger was born on 13 August 1918 in Rendcomb, England, the son of a doctor, and was brought up as a Quaker. He received his PhD at Cambridge in 1943 and stayed in Cambridge for the rest of his career. He died there on 19 November 2013.
He won the 1958 Nobel Prize in Chemistry alone, "for his work on the structure of proteins, especially that of insulin". In 1980 he received a quarter of the Nobel Prize in Chemistry "for their contributions concerning the determination of base sequences in nucleic acids". The 1980 prize was split: half to Paul Berg, and the other half shared by Walter Gilbert and Sanger.
How did he work out the structure of insulin?
In his 1958 Nobel lecture, Sanger says the insulin work took about 12 years. The challenge was to find the order of amino acids in a chain without any way to read it directly.
His first tool was a label. He developed the dinitrophenyl (DNP) method, using the reagent fluorodinitrobenzene, which attaches to the free amino group at the start of a chain. After the protein is broken into amino acids with acid, the labelled one tells you which amino acid came first.
His second tool was breaking chains into overlapping pieces. He split insulin's two chains apart by oxidising the disulphide bridges that hold them together, and found one chain of 30 residues (the B chain) and a shorter A chain. He then broke each chain into short peptides, separated them by chromatography, identified each one, and fitted the overlaps together. The B chain results were published with Hans Tuppy in the Biochemical Journal in 1951. The Nobel page notes that by 1955 he had worked out how the chains are linked.
The lecture adds a striking result: comparing insulins from cattle and four other species, the whole B chain was identical and the only differences were three amino acids inside a disulphide ring of the A chain.
How does dideoxy sequencing read DNA?
In 1977 Sanger, Nicklen and Coulson published DNA sequencing with chain-terminating inhibitors in PNAS. Their abstract describes using dideoxy analogues of the normal DNA building blocks, which "act as specific chain-terminating inhibitors of DNA polymerase".
His 1980 Nobel lecture explains why. A dideoxynucleotide lacks the 3' hydroxyl group. DNA polymerase can add it to a growing chain, but nothing can be attached after it, so the chain stops. Run four reactions, each with a small amount of one dideoxy base (A, C, G or T), and each reaction makes a set of fragments ending at that base. Electrophoresis on a gel sorts the fragments by length.
A worked example: reading a sequencing gel
Suppose a short DNA template gives new strands, counted from the end of the primer, of these lengths:
ddA lane: 2 and 5
ddC lane: 1 and 6
ddG lane: 3
ddT lane: 4
Step 1: list lengths from shortest to longest, with the lane each appears in. 1 (C), 2 (A), 3 (G), 4 (T), 5 (A), 6 (C).
Step 2: read the new strand, 5' to 3'. C A G T A C.
Step 3: get the template. The template is complementary and antiparallel. Pair each base (C with G, A with T) to get G T C A T G, which is the template read 3' to 5'. Written 5' to 3', the template is GTACTG.
Common mistakes: reading the gel from longest to shortest, and forgetting that the gel gives you the newly made strand, not the template.
The lecture describes the payoff. The DNA of bacteriophage phiX174, a single-stranded circle of 5,386 nucleotides coding for ten genes, was sequenced by his copying methods and reported in Nature in 1977. The big surprise was overlapping genes: stretches of DNA that code for two different proteins using different reading frames.
Why did his methods matter so much?
Reading sequence turned biology into something you could check letter by letter. Insulin's structure showed that a protein has one exact sequence. Dideoxy sequencing made it practical to read genes base by base. If you are studying this for NEET or boards, our guides to the molecular basis of inheritance and biotechnology principles connect it to the syllabus.
What can a student take from his way of working?
Build the tool first. Sanger's breakthroughs were methods: a label, a way to break chains, a way to stop copying. Answers followed.
Break a big problem into overlapping small ones. Fragments you can identify, fitted together, beat a whole you cannot.
Stay with a problem. Twelve years on insulin is a reminder that steady work on one question can pay off.
When a step like antiparallel strands keeps tripping you up, the Fermi Concept Library has every concept in biology and chemistry with its own lesson, so you can fix that one idea and return to practice.
Make up your own four-lane gel with eight fragments, swap with a friend, and read each other's sequence and template.
