Sanger Sequencing, also known as Capillary Electrophoresis (CE) sequencing, is a method of DNA sequencing, based on the selective incorporation of dideoxynucleotide chain terminators by DNA polymerase during in vitro DNA replication (cycle sequencing; the DNA polymerase adds four different nucleotides one by one onto a growing DNA template strand in a linear mode. Each incorporated nucleotide is identified by its fluorescent tag (dye). This extension continues until a particular nucleotide is incorporated. Because of the nucleotide’s dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. At the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are then generated. This dye terminator cycle sequencing is the modern variant of the dideoxy chain termination sequencing method first pioneered by Frederick Sanger and colleagues in 1977.
Important aspects in the Sanger Sequencing are the purification (clean-up) steps prior to loading for electrophoresis, and the use of high-quality reagents during all phases of the workflow. This will eliminate any interference, and will optimize conditions to produce high-quality peaks.
Automation of Sanger Sequencing has been made possible with the development of a variety of DNA sequencing instruments and platforms, such as Applied Bioystems® ABI Genetic Analyzers and SeqStudio™, and Promega Spectrum.
Since the introduction of massive parallel sequencing (MPS), or Next-Generation Sequencing (NGS), the Sanger Sequencing method remains in wide use, for smaller-scale projects, validation of NGS results and for obtaining especially long contiguous DNA sequence reads. While the Sanger method only sequences a single DNA fragment at a time, NGS is sequencing millions of fragments simultaneously (parallel) per sequencing run.
