Genetics & Molecular · 1977
Sanger DNA Sequencing (dideoxy chain-termination method)
Dideoxynucleotide chain-termination sequencing
Frederick Sanger had already determined the amino acid sequence of insulin in 1955, work that earned him his first Nobel Prize in Chemistry in 1958. By the mid-1970s, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, he was working on a harder problem: reading the sequence of nucleotides in DNA itself. The challenge was producing fragments of defined lengths that each terminated at a known base, so their size on a gel would directly encode sequence information.
Sanger's solution, published in PNAS in December 1977, used dideoxynucleotides. These modified bases lack the 3'-hydroxyl group needed to extend the growing strand, so when a dideoxynucleotide is incorporated, synthesis stops. By running four separate reactions, each with a different dideoxy terminator, and then separating the resulting nested fragments by size on a polyacrylamide gel, the sequence could be read directly from the banding pattern. In the same year, Allan Maxam and Walter Gilbert at Harvard published a chemical cleavage method that achieved the same goal by a different route. Sanger's approach proved faster, safer to handle, and more scalable.
The 1977 paper shared Sanger a portion of the 1980 Nobel Prize in Chemistry, making him one of only four individuals in history to receive two Nobel Prizes, and the only person to receive two in the same scientific discipline. Gilbert shared the 1980 award; Sanger's Cambridge colleague Paul Berg received the other portion for recombinant DNA work. The back-to-back Nobel recognitions for both protein and nucleic acid sequencing from a single researcher are without parallel in modern biochemistry.
Within a decade, Sanger sequencing had been adapted for fluorescent dye labeling, replacing the radioactive gels of the original protocol, and for capillary electrophoresis, which allowed automation and multiplexing. Applied Biosystems commercialized automated Sanger sequencers in the 1980s. These instruments drove the Human Genome Project, launched in 1990, which produced a working draft of the 3.2-billion base-pair human genome by 2001 and a finished sequence by 2003.
Next-generation sequencing technologies that appeared from the mid-2000s onward offer far higher throughput at lower cost per base, displacing Sanger sequencing for large-scale discovery work. Clinical genetic laboratories continue to use Sanger sequencing to confirm individual variants identified by panel or exome testing, because its per-base accuracy and the interpretive clarity of a single clean trace remain difficult to match when a specific nucleotide position is the question.
Key People
- Frederick Sanger — British biochemist who developed dideoxy sequencing; two-time Nobel laureate (1958, 1980).
- Alan Maxam — Co-developer with Gilbert of the alternative chemical DNA sequencing method, published in parallel.
- Walter Gilbert — Harvard biochemist; shared the 1980 Nobel for the chemical cleavage sequencing method.
- Leroy Hood — Caltech biologist who co-developed the automated fluorescent Sanger sequencer in the 1980s.
Proc Natl Acad Sci USA, 1977
Related landmarks
- 1978 · Recombinant Human Insulin (synthesis of the gene) (Genetics & Molecular)
- 1975 · Southern blot: DNA detection by gel transfer and hybridization (Genetics & Molecular)
- 1985 · Polymerase Chain Reaction (PCR) (Genetics & Molecular)
- 1990 · First Approved Human Gene Therapy (ADA-SCID, Ashanthi DeSilva) (Genetics & Molecular)