Pyrosequencing: Difference between revisions

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Revision as of 07:39, 19 September 2022

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'''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 pyrosequencing.

The principle of pyrosequencing was first described in 1993<ref>Nyren, Pettersson and Uhlen (1993) “Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay” Analytical Biochemistry 208 (1), 171-175, https://doi.org/10.1006/abio.1993.1024</ref> by, [http://kth.diva-portal.org/smash/record.jsf?pid=diva2%3A8081&dswid=3295#sthash.08HyBugr.dpbs Bertil Pettersson], [https://www.kth.se/en/bio/research/proteomics/proteomics-researchers/mathias-uhlen-1.67763 Mathias Uhlen] and [[Pål Nyrén|Pål Nyren]] by combining the [[solid phase sequencing]] method<ref>Uhlen (1989) ”Magnetic separation of DNA” Nature 340: 733-4, https://doi.org/10.1038/340733a0</ref> using [[streptavidin]] coated magnetic beads with recombinant DNA polymerase lacking 3´to 5´exonuclease activity (proof-reading) and luminescence detection using the [[Luciferase|firefly luciferase]] enzyme.<ref>Nyren and Lundin (1985) “Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis” Analytiocal Biochemistry 151 (2): 504-509. https://doi.org/10.1016/0003-2697(85)90211-8</ref> A mixture of three [[enzyme]]s ([[DNA polymerase]], [[Sulfate adenylyltransferase|ATP sulfurylase]] and firefly [[luciferase]]) and a nucleotide ([[Nucleoside triphosphate|dNTP]]) are added to single stranded DNA to be sequenced and the incorporation of nucleotide is followed by measuring the light emitted. 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. The nucleotide mixture is removed before the next nucleotide mixture is added. This process is repeated with each of the four nucleotides until the DNA sequence of the single stranded template is determined.

A second solution-based method for pyrosequencing was described in 1998<ref>{{Cite journal |last1=Ronaghi |first1=Mostafa |last2=Uhlén |first2=Mathias |last3=Nyrén |first3=Pål |date=1998-07-17 |title=A Sequencing Method Based on Real-Time Pyrophosphate |url=https://www.science.org/doi/abs/10.1126/science.281.5375.363 |journal=Science |volume=281 |issue=5375 |pages=363–365 |language=EN |doi=10.1126/science.281.5375.363|pmid=9705713 |s2cid=26331871 }}</ref> by [[Mostafa Ronaghi]], [https://www.kth.se/en/bio/research/proteomics/proteomics-researchers/mathias-uhlen-1.67763 Mathias Uhlen] and [[Pål Nyrén|Pål Nyren]]. In this alternative method, an additional enzyme [[apyrase]] is introduced to remove nucleotides that are not incorporated by the DNA polymerase. This enabled the enzyme mixture including the [[DNA polymerase]], the [[luciferase]] and the [[apyrase]] to be added at the start and kept throughout the procedure, thus providing a simple set-up suitable for automation. An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.

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==Commercialization==

The company '''Pyrosequencing AB''' 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. Pyrosequencing AB was listed on the [[Stockholm Stock Exchange]] in 1999. It was renamed to [httpBiotage in 2003.<ref>{{Cite web |last=Biotage |title=Biotage History |url=https://www.biotage.com/biotage-history Biotage]|access-date=2022-09-19 in 2003|website=www.biotage.com |language=en}}</ref> The pyrosequencing business line was acquired by [[Qiagen]] in 2008. Pyrosequencing technology was further licensed to [[454 Life Sciences]]. 454 developed an array-based pyrosequencing technology which emerged as a platform for [[DNA sequencing#High-throughput sequencing|large-scale DNA sequencing]], including [[genome project|genome sequencing]] and [[metagenomics]].

[[Hoffmann-La Roche|Roche]] announced the discontinuation of the 454 sequencing platform in 2013.<ref>{{cite news|last1=Hollmer|first1=Mark|title=Roche to close 454 Life Sciences as it reduces gene sequencing focus |url=http://www.fiercebiotech.com/medical-devices/roche-to-close-454-life-sciences-as-it-reduces-gene-sequencing-focus|work=Fierce Biotech|date=October 17, 2013}}</ref>