Figure 1.

 Pyrosequencing technology. A sequencing primer is hybridized to a single‐stranded, PCR‐amplified DNA template and incubated with a DNA polymerase. Each event of incorporation of a dNTP is accompanied by the release of pyrophosphate (PPi) in a quantity equimolar to the amount of nucleotide incorporated. This PPi is quantitatively converted to ATP by ATP sulfurylase in the presence of adenosine 5′‐phosphosulfate. The ATP thus obtained drives the luciferase‐mediated conversion of luciferin to oxyluciferin, thus generating visible light in amounts proportional to the amount of ATP generated. The light produced in this luciferase‐catalyzed reaction is detected by a charge‐coupled device (CCD) camera and seen as a peak in a pyrogram. Apyrase – a nucleotide‐degrading enzyme – continuously degrades unincorporated dNTPs and excess ATP, during synthesis of the complementary DNA. Gray columns indicate quantified CpG sites.