How does pyrosequencing work?

Pyrosequencing is a DNA sequencing method based on detecting the chemiluminescent signal emitted during the sequential addition of nucleotides. There are four major reactions involved in pyrosequencing, which are catalyzed four different enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase. Two substrates, adenosine 5´ phosphosulfate (APS) and luciferin, are also required in this process.

• One of the four dNTPs is added to the growing DNA strand by DNA polymerase, during which pyrophosphate (PPi) is released. Since dATP is a substrate for a luciferase, which may increase the noise signal, dATP?S is used in this step.
• PPi is converted to ATP by ATP sulfurylase in the presence of adenosine 5´ phosphosulfate.
• Luciferin is converted to oxyluciferin with ATP as a second substrate. This reaction generates the visible chemiluminescent signal, which is detected by a camera and analyzed in a program.
• Unincorporated nucleotides and ATP are degraded by the apyrase. The reaction can restart with another nucleotide.