Pyrosequencing

Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released, hence, the name given it.

Principles

The principle of pyrosequencing was first described in 1993 by P. Nyrén, B. Pettersson, and M. Uhlen.^{[ non-primary source needed ] [1]} The technique combines solid phase sequencing, and use of streptavidin-coated magnetic beads, a recombinant DNA polymerase lacking 3´-to-5´exonuclease activity (proof-reading), and luminescence detection of inorganic pyrophosphate using the firefly luciferase enzyme.^{[ non-primary source needed ] [2] [3] [ clarification needed ]}

Specifically, a solution of three enzymes—DNA polymerase, ATP sulfurylase, and firefly luciferase—and a deoxyribonucleoside triphosphate (dNTP) are added to single stranded DNA to be sequenced, and the incorporation of nucleotide is followed, measuring the light emitted as a consequence of inorganic pyrophosphate production.^{[ citation needed ]} The intensity of the light determines if 0, 1, or more nucleotides have been incorporated, thus showing how many complementary nucleotides are present on the template strand.^{[ citation needed ]} The nucleotide mixture is removed before a next nucleotide mixture is added, and the process is repeated for each of the four nucleotides, until the DNA sequence of the single stranded template is determined.^{[ citation needed ]}

A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi, [Mathias Uhlen], ^[4] and Pål Nyren.^{[ non-primary source needed ] [5]} In this alternative method, an additional enzyme, apyrase, is introduced to remove nucleotides that are not incorporated by the DNA polymerase.^{[ citation needed ]} This enables the enzyme mixture— DNA polymerase, luciferase, and apyrase—to be added when sequencing is initiated, and kept in the reaction solution throughout the procedure (thus enabling easier automation).^{[ citation needed ]} An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.^{[ citation needed ]}

A third variant, a microfluidic pyrosequencing method, was described in 2005 by an industrial research team led by Jonathan Rothberg, at the company 454 Life Sciences.^{[ non-primary source needed ] [6]} This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support; Rothberg and co-workers demonstrated that sequencing could be performed in a highly parallel manner using a microfabrication and microarrays.^{[ citation needed ]} This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.^{[ citation needed ]} This first next generation sequencing instrument initiated a new era in genomics research,^{[ according to whom? ]} and to rapidly falling prices for DNA sequencing,^{[ according to whom? ]} allowing affordable whole genome sequencing.^{[ citation needed ]}

Procedure

The chart shows how pyrosequencing works.

"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template. ^{[7] [ better source needed ][ verification needed ]}

For the solution-based version of pyrosequencing, the single-strand DNA (ssDNA) template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin. ^{[7] [ verification needed ]}

1. The addition of one of the four deoxynucleotide triphosphates initiates the second step; dNTPs)—dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi). ^{[7] [ verification needed ]}

1. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount.^{[ clarification needed ]} The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program. ^{[7] [ verification needed ]}

1. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide. ^{[7] [ verification needed ]}

The process can be represented by the following equations:

• PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase);
• ATP + luciferin + O2 → AMP + PPi + oxyluciferin + CO₂ + hv (catalyzed by luciferase);

where PPi is pyrophosphate, APS is adenosine 5-phosphosulfate, ATP is adenosine triphosphate, O2 is dioxygen, AMP is adenosine monophosphate, CO₂ is carbon dioxide, and hv is light. ^{[7] [ verification needed ]}

Limitations

Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing).^{[ citation needed ]} This can make the process of genome assembly more difficult, particularly for sequences containing a large amount of repetitive DNA.^{[ citation needed ]} Also, lack of a proof-reading activity^{[ clarification needed ]} limits accuracy of this method.^{[ citation needed ]}

Commercialization

Pyrosequencing AB, a company based in Uppsala, Sweden, was founded with venture capital provided by HealthCap in order to commercialize machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique. ^{[8] [ verification needed ]} Pyrosequencing AB was listed on the Stockholm Stock Exchange in 1999. ^{[8] [ verification needed ]} When Pyrosequencing AB acquired Biotage LLC, a U.S.-based company, and other companies, in 2003, the company was renamed Biotage AB. ^[8] The pyrosequencing and other biomedical units of Biotage AB were sold to Qiagen in 2008. ^{[8] [ verification needed ]} The pyrosequencing technology was licensed to 454 Life Sciences.^{[ when? ][ citation needed ]} 454 developed an array-based pyrosequencing technology that emerged as a platform for large-scale DNA sequencing, including genome sequencing and metagenomics.^{[ citation needed ]}

Roche acquired 454 Life Sciences,^{[ when? ][ citation needed ]} and announced the discontinuation of the 454 sequencing platform in 2013. ^[9] The 454 sequencing platform was replaced, in part, by Illumina dye sequencing, and by Applied Biosystems sequencing products.^{[ citation needed ]}

Further reading

• Harrington, C.T.; Lin, E.I.; Olson, M.T. & Eshleman, J.R. (1 September 2013). "Fundamentals of Pyrosequencing". Arch. Pathol. Lab. Med. 137 (9): 1296–1303. doi:10.5858/arpa.2012-0463-RA . Retrieved 29 October 2025. For the corresponding PDF document, see this link.

• Metzker M. (2005). "Emerging Technologies in DNA Sequencing". Genome Research. 15 (12): 1767–76. doi: 10.1101/gr.3770505 . PMID   16339375.

References

1. Nyrén, P.; Pettersson, B. & Uhlen, M. (January 1993). "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay" . Analytical Biochemistry. 208 (1): 171–175. doi:10.1006/abio.1993.1024. ISSN   0003-2697 . Retrieved 28 October 2025.^{[ non-primary source needed ]}
2. Uhlen, M. (31 August 1989). "Magnetic Separation of DNA" . Nature (London) . 340: 733–734. doi:10.1038/340733a0 . Retrieved 28 October 2025.^{[ non-primary source needed ]}
3. Nyrén, Pål & Lundin, Arne (December 1985). "Enzymatic Method for Continuous Monitoring of Inorganic Pyrophosphate Synthesis" . Analytical Biochemistry. 151 (2): 504–509. doi:10.1016/0003-2697(85)90211-8. ISSN   0003-2697 . Retrieved 28 October 2025.^{[ non-primary source needed ]}
4. https://www.kth.se/en/bio/research/proteomics/proteomics-researchers/mathias-uhlen-1.67763
5. Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (17 July 1998). "A Sequencing Method Based on Real-Time Pyrophosphate" . Science. 281 (5375): 363–365. doi:10.1126/science.281.5375.363. PMID   9705713. S2CID   26331871.^{[ non-primary source needed ]}
6. Margulies, M.; Egholm, M.; Altman, W.E.; Attiya, S.; Bader, J.S.; Bemben, L.A.; Berka, .; Braverman, M.S.; Chen, Y.-J.; Chen, Z.; Dewell, S.B.; Du, L.; Fierro, J.M.; Gomes, X.V.; Godwin, B.C.; He, W.; Helgesen, S.; Ho, C.H.; Irzyk, G.P.; Jando, S.C.; Alenquer, M.L.I.; Jarvie, T.P.; Jirage, K.B.; Kim, J.-B.; Knight, J.R.; Lanza, J.R.; Leamon, J.H.; Lefkowitz, S.M.; Lei, M.; Li, J.; Lohman, K.L.; Lu, H.; Makhijani, V.B.; McDade, K.E.; McKenna, M.P.; Myers, E.W.; Nickerson, E.; Nobile, J.R.; Plant, R.; Puc, B.P.; Ronan, M.T.; Roth, G.T.; Sarkis, G.J.; Simons, J.F.; Simpson, J.W.; Srinivasan, M.; Tartaro, K.R.; Tomasz, A.; Vogt, K.A.; Volkmer, G.A.; Wang, S.H.; Wang, Y.; Weiner, M.P.; Yu, P.; Begley, R.F. & Rothberg, J.M. (31 July 2005). "Genome Sequencing in Microfabricated High-Density Picolitre Reactors" . Nature (London) . 437: 376–380. doi:10.1038/nature03959 . Retrieved 28 October 2025.^{[ non-primary source needed ]}
7. Qiagen Staff (4 August 2017). "Pyrosequencing Technology and Platform Overview". Qiagen Knowledge & Support/Knowledge Hub/Technology & Research/Pyrosequencing Resource Center (Qiagen.com). Retrieved 4 August 2017.^{[ better source needed ]}
8. Biotage Staff. "Biotage History". Biotage.com. Retrieved 19 September 2022.
9. Hollmer, Mark (17 October 2013). "Roche to Close 454 Life Sciences as it Reduces Gene Sequencing Focus". Fierce Biotech . Retrieved 28 October 2025.