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How did the Sanger method sequence DNA?

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Diagram of a DNA double helix showing its twisted ladder structure, with a close-up of the sugar-phosphate backbone, paired nitrogenous bases (adenine, thymine, cytosine, guanine), and labeled nucleotides and hydrogen bonds. — human genome The human genome is made up of approximately three billion base pairs of deoxyribonucleic acid (DNA). The bases of DNA are adenine (A), thymine (T), guanine (G), and cytosine (C).

The Sanger method, also known as the dideoxy method, was a foundational first-generation technology used to determine the nucleotide sequence of DNA.12

The process works by synthesizing new DNA chains on a template strand. During synthesis, the reaction incorporates four possible dideoxy nucleotides, which lack a 3' hydroxyl group. Because this group is necessary for adding subsequent nucleotides, its absence causes chain growth to stop whenever a dideoxy nucleotide is incorporated.12

This procedure generates a population of nested, truncated DNA molecules, with each fragment representing the site of a specific nucleotide in the template. These fragments are then separated by size using electrophoresis. In later iterations, the method was automated using fluorescent tags, where fragments were separated in glass capillaries and detected via laser excitation. Finally, the nucleotide sequence is deduced using a computer.12