DNA sequencing with chain-terminating inhibitors¶
Why this mattered¶
Sanger, Nicklen, and Coulson’s 1977 paper turned DNA sequencing from a specialized, fragile procedure into a broadly usable experimental method. Its key shift was the use of chain-terminating nucleotide analogues: DNA polymerase would copy a template until occasional incorporation of a dideoxynucleotide stopped extension at a specific base. By separating the resulting fragments, researchers could read sequence directly from ordered termination products. Compared with the earlier “plus and minus” method, this made sequencing faster, more accurate, and more scalable, as the authors demonstrated on bacteriophage φX174 DNA.
What became newly possible was the routine determination of primary genetic information. DNA sequence could now be treated as an experimental object in its own right, not merely inferred from genetics, restriction maps, or protein products. This enabled complete viral and organelle genomes, systematic gene characterization, mutation analysis at single-nucleotide resolution, and eventually the construction of large sequencing projects. The method’s logic also fit naturally with later automation: fluorescent labels, capillary electrophoresis, and computer base-calling preserved the same chain-termination principle while replacing radioactive gels and manual reading.
The paper therefore sits at the root of modern genomics. The Human Genome Project, clinical variant discovery, comparative genomics, and much of molecular evolution depended on the ability to produce reliable DNA sequence at scale, and Sanger sequencing was the dominant foundation for that work for decades. Even after next-generation sequencing transformed throughput and cost, chain-termination sequencing remained a reference standard for targeted validation because of its accuracy and clear read structure. Its importance was not only that it improved an existing technique, but that it made the sequence of DNA a practical, central measurement in biology.
Abstract¶
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Related¶
- enables → A novel potent vasoconstrictor peptide produced by vascular endothelial cells — Sanger sequencing enabled identification of the amino-acid sequence of endothelin, the endothelial vasoconstrictor peptide reported in 1988.
- enables → The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 — Sanger chain-termination sequencing enabled the nucleotide-level identification of lin-4 small RNAs and their complementarity to lin-14.
- enables → Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors — Sanger chain-termination sequencing enabled determination of the nucleotide sequences of the M13mp18 and pUC19 cloning vectors.
- enables → The Sequence of the Human Genome — Sanger chain-termination sequencing enabled the base-calling technology used to generate the Human Genome Project's reference sequence.
- enables → Positional cloning of the mouse obese gene and its human homologue — Sanger chain-termination sequencing enables obese-gene cloning by making nucleotide-level identification of the mouse gene and human homologue possible.
- enables → Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase — Sanger chain-termination sequencing enabled PCR development by establishing primer-directed DNA polymerase extension as a controllable method for copying specific DNA.
- cite ← A novel potent vasoconstrictor peptide produced by vascular endothelial cells — The endothelin paper used Sanger chain-termination DNA sequencing to determine the nucleotide sequence encoding the peptide precursor.
- cite ← The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 — The lin-4 paper uses Sanger chain-termination sequencing to identify the small lin-4 RNA transcripts.
- cite ← Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors — The M13 vector paper supports Sanger chain-termination sequencing by providing improved single-stranded phage templates and vector sequences.
- cite ← The Sequence of the Human Genome — The human genome paper depends on Sanger chain-termination sequencing as the core technology enabling reference genome assembly.
- cite ← Positional cloning of the mouse obese gene and its human homologue — The obese-gene cloning study uses Sanger chain-termination sequencing to determine the mouse obese gene and human homolog sequences.
- cite ← Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase — Thermostable PCR amplification enabled efficient preparation of DNA templates for Sanger chain-termination sequencing.