File:A-Primer-on-Metagenomics-pcbi.1000667.g002.jpg

English: Single stranded DNA template is first hybridized with the sequencing primer and mixed with the enzymes along with the two substrates adenosine 5′-phosphosulfate (APS) and luciferin. In each cycle, (1) one of the four nucleotides (dTTPi, in this case) is then added to the reaction. (2) If the nucleotide is complementary to the base in the template strand then the DNA polymerase incorporates it into the growing strand. (3) Pyrophosphate (PPi)—in an amount equal in molarity to that of the incorporated nucleotide—is released and converted to ATP by sulfurylase in the presence of APS. (4) ATP then serves as a substrate to luciferase, causing a light reaction. Photon emission is in equimolar quanta to the amount of nucleotide incorporated in a given cycle. (5) The excess nucleotides are degraded by apyrase.