1951

Sanger Sequencing

Archer John Porter Martin (1910–2002), Richard Laurence Millington Synge (1914–1994), Frederick Sanger (1918–2013), Hans Tuppy (b. 1924)

Present-day chemists and molecular biologists are accustomed to manipulating proteins with confidence, figuring out their amino acid sequences and producing variations at will. But before 1951 the structures of proteins was a mystery. With twenty amino acid options per position, the number of possible proteins gets out of control very, very quickly, and there were many important open questions. Did a given protein always have a particular sequence and a particular structure? Perhaps just the active sites of a protein were fixed, and the rest of it could vary? No one would know until amino acid sequences could be determined with certainty.

In 1951, Sanger and coworkers, in particular Austrian biochemist Hans Tuppy, showed the world how it could be done, sequencing the “B chain” of insulin. They were helped by the revolutionary 1943 discovery by English chemist Archer John Porter Martin and English biochemist Richard Laurence Millington Synge that amino acids, small peptides, and other molecules could be separated by chromatography, letting the solvent soak upward from a mixture spotted near the bottom of a sheet of filter paper.

Sanger invented a reaction that would make a brightly colored dinitrophenyl (DNP) derivative out of the amino end of a peptide chain (the end of the chain with a free amine, or NH₂, group) without disturbing the rest of it, but then stay attached even after the amino acids were broken apart. Sequencing of short peptides was now possible by working backward from the amino end, albeit with painstaking work and with allowance for amino acids that reacted outside the usual patterns. For insulin, Sanger and his team broke up the protein into an assortment of shorter pieces. These were separated and run through the sequencing scheme, and then the whole protein was pieced together like a puzzle.

The result proved that proteins do have specific sequences that give them their shapes and properties—a critical advance that won Sanger a Nobel and suggested that cells must somehow contain exact codes for all their proteins. Sanger later invented techniques to sequence DNA and RNA, and won a second Nobel for that!

A molecular model of the insulin protein. Sanger’s methods not only provided the composition of a protein, but they also helped chemists to think of them as actual organic compounds that could be studied and manipulated using chemical techniques.