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Highly sensitive pyrosequencing based on the capture of free APS with ATP sulfurylase.
Anal Chem. 2011 Mar 25;
Authors: Wu H, Wu W, Chen Z, Wang W, Zhou G, Kajiyama T, Kambara H
In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, ATP sulfurylase, luciferase and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes photo emission reaction. However the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence the sensitivity cannot be much higher due to the simultaneous increase of the background signal when using a larger amount of luciferase. In this study, we demonstrated a sensitive pyrosequencing by using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode-array for constructing a portable pyrosequencer, a potential tool in point-of-care test (POCT).
PMID: 21438613 [PubMed - as supplied by publisher]
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Highly sensitive pyrosequencing based on the capture of free APS with ATP sulfurylase.
Anal Chem. 2011 Mar 25;
Authors: Wu H, Wu W, Chen Z, Wang W, Zhou G, Kajiyama T, Kambara H
In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, ATP sulfurylase, luciferase and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes photo emission reaction. However the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence the sensitivity cannot be much higher due to the simultaneous increase of the background signal when using a larger amount of luciferase. In this study, we demonstrated a sensitive pyrosequencing by using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode-array for constructing a portable pyrosequencer, a potential tool in point-of-care test (POCT).
PMID: 21438613 [PubMed - as supplied by publisher]
More...