DNA is sequenced enzymatically using ABI's Big Dye Terminator chemistry. One primer and one template are mixed with DNA polymerase, buffer, Mg2+ deoxynucleotides, and dideoxynucleotides with specific fluorescent dyes attached to each base.
Reactions are carried out in a thermal cycler, cycling between denaturation (96°C), annealing (50°C) and extension (60°C) temperatures for 25 cycles. The DNA fragments with a dye labeled termination electrophorese through the polymer in the capillary, separating fragments by molecular weight. The DNA passes a detection window at the end of the capillary, where the fluorescent dyes are excited with a laser beam and emit light at wavelengths unique to each terminator.
A charged-coupled device (CCD) camera captures the emitted light, converts it to electronic information, and then sends it off to a computer. The computer analyzes the signal to generate sequence data and electropherograms. The computer generated sequence and electropherograms are available via the web-ordering site.