Figure 1.

Automated ultrahigh-throughput parallel pyrosequencing. A single strand of each DNA fragment to be sequenced (a maximum of around 500 bp long) is amplified by PCR to make a single-strand DNA library and immobilized as one strand per bead. (a) Each bead is captured in its own microchamber and the DNA amplified by PCR, resulting in a bead coated with millions of copies of this single strand. (b) For sequencing, the DNA-coated beads are placed into a plate containing more than a million individual fiber-optic picoliter-scale wells, such that each well contains no more than one bead, and all the wells are then sequenced simultaneously by proprietary pyrosequencing technology. Pyrosequencing is based on primer-initiated DNA synthesis on the single strands, using repeated cycles of each of the four nucleotides (added as pyrophosphates) in turn. Addition of a base (or more than one base at a time) to the growing strand is accompanied by release of pyrophosphate and the consequent activation of luciferin and a flash of light, which is recorded via the fiber optics (the intensity of the flash correlates with the number of bases added). The light generation system depends on the reaction of pyrophosphate with APS [23] catalyzed by sulfurylate to give ATP. The luciferase catalyzed reaction of ATP and luciferin generates a flash of light. The sequence of the DNA in each well is then recovered from the recorded data. Figure reproduced with permission from [23].