- Sanger sequencing, also known as the chain-termination method, is a DNA Sequencing technique developed by Frederick Sanger and his colleagues in 1977. It revolutionized the field of molecular biology by enabling researchers to determine the precise nucleotide sequence of DNA molecules.
- The method is based on the selective incorporation of chain-terminating dideoxyribonucleotides (ddNTPs) during DNA replication. These ddNTPs lack a 3′ hydroxyl group, which prevents the addition of further nucleotides once incorporated, effectively terminating DNA strand elongation at specific bases. By incorporating a mixture of normal deoxyribonucleotides (dNTPs) and fluorescently labeled ddNTPs, DNA fragments of varying lengths are generated, each ending with a labeled terminator corresponding to a specific nucleotide.
- The Sanger sequencing process begins with the denaturation of the double-stranded DNA template, followed by the annealing of a short primer that binds to a complementary region near the sequence of interest. DNA polymerase is then added to extend the primer by adding complementary nucleotides to the growing DNA strand. During this synthesis reaction, small amounts of each of the four ddNTPs are included in the reaction mixture alongside the normal dNTPs. When a ddNTP is incorporated instead of a dNTP, the elongation of that DNA strand ceases. Because this occurs randomly at each occurrence of a particular base, a population of DNA fragments of different lengths is produced, each ending in a known base.
- To resolve these fragments and identify the terminal nucleotide, the mixture is subjected to capillary electrophoresis. This technique separates the DNA fragments by size with high resolution. The fluorescent labels attached to the ddNTPs are detected by a laser as the fragments pass through a detector, allowing automated systems to record the sequence based on the color emitted by each labeled base. The output is a chromatogram with colored peaks corresponding to the four bases, enabling the reconstruction of the original DNA sequence.
- Although high-throughput next-generation sequencing (NGS) methods have largely supplanted Sanger sequencing for large-scale genomic projects, Sanger sequencing remains the gold standard for small-scale sequencing tasks due to its accuracy, simplicity, and relatively long read lengths (up to 800–1000 base pairs). It is widely used for sequencing single genes, confirming mutations identified by other methods, validating NGS data, and in applications such as genotyping, mutation analysis, and microbial identification.
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