Highly Sensitive Pyrosequencing Based on the Capture of Free Adenosine 5′ Phosphosulfate with Adenosine Triphosphate Sulfurylase

摘要

In pyrosequencing chemistry, four cascade enzymaticnreactions with the catalysis of polymerase, adenosinentriphosphate (ATP) sulfurylase, luciferase, and apyrase arenemployed. The sensitivity of pyrosequencing mainly dependsnon the concentration of luciferase which catalyzes a photoemissionnreaction. However, the side-reaction of adenosine 50nphosphosulfate (APS, an analogue of ATP) with luciferasenresulted in an unavoidable background signal; hence, thensensitivity cannot be much higher due to the simultaneousnincrease of the background signal when a larger amount ofnluciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower thenconcentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase,na large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencingnchemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential toolnin a point-of-care test (POCT).