How does Sanger sequencing work?

Automated fluorescent DNA sequencing using a capillary DNA sequencing instrument (such as the ABI 3130XL Genetic Analyzer in the UD S&GC) is based on the use of a different colored fluorescent dye for each of the four DNA bases. Linking these dyes to the four dideoxynucleotide terminators utilized in the Sanger chain-termination DNA sequencing reaction results in all fragments ending in one of the four fluorescent dye-labeled terminator corresponding to the dideoxy bases. These fragments are then separated by electrophoresis through a flowable polymer pumped into each capillary. The fluorescence is detected by laser excitation as the dye-labeled fragments migrate through a transparent window of the capillary array, this detector window sits in front of a CCD camera. The use of four different dyes allows the sequencing reaction to be performed in a single tube and the resulting dye-labeled terminator fragments to be injected into a single capillary. To view the sequencing process in action I recommend the Macromedia Flash animation at the Dolan DNA Learning Center.

Image of raw data from 16 different samples being captured and recorded by the CCD camera.

Image of analyzed sequence data, commonly referred to as an electropherogram or trace file.