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How does DNA sequencing determine the order of bases in a DNA molecule?

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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).

DNA sequencing determines the order of nucleotides in a DNA molecule by measuring the distance from a fixed point to each occurrence of a specific base (A, C, G, or T).1

Early methods, such as the Maxam-Gilbert method and the Sanger method, relied on creating DNA fragments of varying lengths. In the Sanger approach, chain growth is terminated at specific nucleotides using dideoxy nucleotides, producing a population of truncated molecules. These fragments are separated by size using gel electrophoresis or capillary electrophoresis, allowing the sequence to be read. Automated versions of this process use fluorescent labels and laser detection to identify the bases.12

Modern next-generation sequencing technologies have largely replaced these first-generation methods. These systems are massively parallel, enabling the simultaneous sequencing of millions of DNA fragments. This high-throughput capacity, supported by advancements in bioinformatics, allows for the rapid and cost-efficient analysis of entire genomes.12