DNA sequencing with chain-terminating inhibitors

Before the development of efficient sequencing techniques, determining the precise order of nucleotides in DNA was a laborious and slow process. Previous methods, such as the 'plus and minus' technique developed by the authors, relied on complex enzymatic reactions that were often difficult to interpret and prone to errors. The goal was to find a more direct, rapid, and reliable way to read the genetic code.

The authors present a novel approach that utilizes 2',3'-dideoxy and arabinonucleoside analogues of normal deoxynucleoside triphosphates. In this method, these analogues act as specific chain-terminating inhibitors. When DNA polymerase attempts to synthesize a new DNA strand, the inclusion of these modified nucleotides prevents the addition of any further nucleotides, effectively 'terminating' the chain at a specific base. By running these reactions in the presence of these inhibitors, researchers can generate a collection of DNA fragments of varying lengths, each ending at a known base. These fragments can then be separated by size to reveal the exact sequence of the original DNA template.

This technique, famously applied to the bacteriophage phiX174, proved to be significantly faster and more accurate than its predecessors. By simplifying the biochemical requirements for sequencing, the dideoxy method (often called Sanger sequencing) became the foundational technology for DNA analysis for decades, enabling the eventual mapping of complex genomes and revolutionizing molecular biology.