Editing Pyrosequencing (section)

{{Short description|Method of DNA sequencing}}
'''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, Bertil Pettersson, [[Mathias Uhlén|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 |url-access=subscription }}</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.

A third microfluidic variant of the pyrosequencing method was described in 2005<ref>Marguiles et al (2005) “Genome sequencing in microfabricated high-density picolitre reactors” Nature 437, 376-380.  https://doi.org/doi:10.1038/nature03959;</ref> by [[Jonathan Rothberg]] and co-workers at the company [[454 Life Sciences]]. This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support and they showed that sequencing could be performed in a highly parallel manner using a [[Microfabrication|microfabricated]] [[microarray]]. This allowed for high-throughput DNA sequencing and an automated instrument was introduced to the market. This became the first next generation sequencing instrument starting a new era in [[genomics]] research, with rapidly falling prices for [[DNA sequencing]] allowing [[whole genome sequencing]] at affordable prices.