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How does pyrosequencing work?
Posted July 22, 2020
Answer
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.
Additional resources
Helixyte™ Green *10,000X Aqueous PCR Solution*
6-ROX glycine *25 uM fluorescence reference solution for PCR reactions* Krishna, B. M., Khan, M. A., & Khan, S. T. (2019). Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence Analysis. In Microbial Genomics in Sustainable Agroecosystems (pp. 89-109). Springer, Singapore. Ju, J., Kim, D. H., Bi, L., Meng, Q., Bai, X., Li, Z., ... & Edwards, J. R. (2006). Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proceedings of the National Academy of Sciences, 103(52), 19635-19640.