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Labeled dideoxynucleotide termination of DNA strand growth, pioneered by Nobel laureate Frederick Sanger, has become the cornerstone of genomics. The ABI 3730 Genetic Analyzer utilizes lasers to illuminate the fluorescent dyes on the terminated strand. Single-reaction sequencing has expanded to massively parallel sequencing [a.k.a. next-generation sequencing (NGS)]. Illumina’s platforms use the same technology, but the fluorescence is read directly through a sensitive light meter. Thermofisher’s NGS technology now has a silicon chip with millions of pH meters built in, but doesn’t measure chain termination. Instead, it measures a H+ release from a growing strand, which is very different from Sanger sequencing. Although powerful, NGS covers full genomes or large DNA populations. Many researchers still need to focus on a small set of genes, thus Sanger sequencing on single targets is still a very viable technology in the lab, including clinical work.

Utilizing a single target and a single primer, fluorescent dideoxynucleotides terminate a growing strand of DNA in a thermal cycling reaction. After washing, the DNA population from the cycle sequencing reaction is separated by size through capillary electrophoresis, then the dideoxynucleotides are illuminated with a laser.