DNA sequencing is a laboratory technique for determining the exact order of nucleotides in a DNA molecule, revealing the linear arrangement of adenine, guanine, cytosine and thymine.
Historical Development and Technologies
First-generation sequencing technologies emerged in the late 1970s and included the chemical degradation method of Maxam and Gilbert and the chain‑termination method devised by Frederick Sanger【189723618703026†L247-L255】. In Sanger’s technique, DNA synthesis is carried out on a template strand, but incorporation of a dideoxynucleotide lacking a 3′ hydroxyl group prevents further elongation, generating populations of nested, truncated molecules【189723618703026†L252-L257】. The fragments are separated by electrophoresis, and the sequence is deduced either by comparing radiolabeled bands or, in automated systems, by detecting fluorescent tags in thin capillaries【189723618703026†L258-L261】. Next‑generation platforms introduced in the 2000s use massively parallel reactions to sequence millions of fragments simultaneously, dramatically increasing throughput and reducing cost【189723618703026†L266-L271】. These methods include sequencing by synthesis (Illumina), pyrosequencing, and semiconductor sequencing. Advances in bioinformatics were critical for managing the large datasets produced by high‑throughput instruments【189723618703026†L266-L271】. Third‑generation approaches, such as nanopore and single‑molecule real‑time sequencing, read long DNA molecules directly without amplification, allowing rapid assembly of complex genomes. DNA sequencing underpins modern microbiology by enabling whole‑genome analysis, comparative genomics, metagenomics, and surveillance of emerging pathogens.
Applications and Examples
Sanger sequencing was used to determine the first complete genomes, such as bacteriophage \u03bb and the human mitochondrial genome. Routine use of this method in diagnostic laboratories continues for verifying small inserts, plasmids and genetic variants. Next‑generation sequencing allows researchers to sequence entire bacterial or viral genomes in a single run, which is vital for tracking antibiotic resistance genes and monitoring outbreaks such as those caused by Salmonella or SARS‑CoV‑2. Sequencing of the 16S ribosomal RNA gene has become a standard tool for identifying bacteria and reconstructing microbial phylogeny. Metagenomic sequencing analyzes DNA from environmental samples to study uncultivable microorganisms. In clinical settings, targeted gene panels and exome sequencing aid in diagnosing inherited disorders and guiding cancer therapy.
The capacity to read DNA sequences has transformed biological research and medicine by providing precise genomic information. As sequencing technologies continue to evolve, costs decline and data quality improves, enabling more comprehensive studies of genetic variation and microbial diversity.
Related Terms: Sanger sequencing, Next-generation sequencing, Polymerase chain reaction, Genome, Bioinformatics