SangerSequencing

Sanger sequencing, also known as dideoxy chain-termination sequencing, is a method of determining the nucleotide sequence of DNA. Developed by Frederick Sanger and colleagues in 1977, it was the first widely used DNA sequencing technology and laid the foundation for modern genomics.

The method uses DNA polymerase to synthesize a complementary strand from a template, in the presence of normal deoxynucleotides and a small proportion of chain-terminating dideoxynucleotides ddNTPs labeled with dyes. When a ddNTP is incorporated, synthesis terminates at that base, generating fragments of varying lengths. A detector then reads the terminal base of each fragment to determine the sequence.

Historically, the method enabled rapid sequencing and was refined with automated fluorescence detection in the late 1980s. It became a workhorse for plasmid verification, gene sequencing, and targeted analyses before the rise of high-throughput next-generation sequencing.

Applications include sequencing of individual genes or small genomes, mutation detection, and quality control in cloning projects. Modern capillary-based Sanger sequencing can read individual fragments of roughly 700 to 1,000 bases.

Limitations include relatively low throughput and higher cost per base compared with next-generation technologies, and shorter usable read lengths in some contexts. Nevertheless, Sanger sequencing remains a reliable, accurate method for specific applications and is commonly used to validate NGS results or sequence simple constructs.