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{{Short description|Method of DNA sequencing}} | {{Short description|Method of DNA sequencing}} | ||
'''Pyrosequencing''' is a | {{use dmy dates|date=October 2025}} | ||
'''Pyrosequencing''' is a non-electrophoretic [[DNA sequencing]] (determining the order of [[nucleotides]] in DNA) method 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. | |||
The principle of pyrosequencing was first described in 1993<ref> | ==Principles<!-- I would transform this section in "History", imrpoving the chronology of discovery and commercialization of the method, while the detailed principle can go in the next session. -->== | ||
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{{refimprove section|date=October 2025}} | |||
{{primary sources|section|date=October 2025}} | |||
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The principle of pyrosequencing was first described in 1993 by [[Pål Nyrén|P. Nyrén]], B. Pettersson, and [[Mathias Uhlén|M. Uhlen]].{{primary source inline|date=October 2025}}<!--ONE CANNOT ESTABLISH A DISCOVERY FROM THE PRIMARY SOURCES OF A SUGGESTED DISCOVERER. SECONDARY SOURCES ARE ABSOLUTELY REQUIRED.--><ref name=":0">{{Cite journal |last1=Ahmadian |first1=Afshin |last2=Ehn |first2=Maria |last3=Hober |first3=Sophia |date=2006 |title=Pyrosequencing: History, biochemistry and future |url=https://linkinghub.elsevier.com/retrieve/pii/S0009898105004274 |journal=Clinica Chimica Acta |language=en |volume=363 |issue=1–2 |pages=83–94 |doi=10.1016/j.cccn.2005.04.038|pmid=16165119 |url-access=subscription }}</ref><ref name = NyrenPetterssonUhlen1993>{{cite journal | author = Nyrén, P.; Pettersson, B. & Uhlen, M. | date = January 1993 | title = Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay | journal = Analytical Biochemistry | volume = 208 | issue = 1 | pages = 171–175 | issn = 0003-2697 | url = https://www.sciencedirect.com/science/article/pii/S0003269783710249 | access-date = 28 October 2025 | url-access = subscription | doi = 10.1006/abio.1993.1024 | pmid = 8382019 }}{{primary source inline|date=October 2025}}</ref><ref name=":1">{{Cite journal |last1=Ghemrawi |first1=Mirna |last2=Tejero |first2=Nicole Fernandez |last3=Duncan |first3=George |last4=McCord |first4=Bruce |date=2023 |title=Pyrosequencing: Current forensic methodology and future applications—a review |url=https://onlinelibrary.wiley.com/doi/abs/10.1002/elps.202200177 |journal=Electrophoresis |language=en |volume=44 |issue=1–2 |pages=298–312 |doi=10.1002/elps.202200177 |pmid=36168852 |issn=1522-2683|url-access=subscription }}</ref><!--Who was corresponding author? Also, no full names to appear until a source is presented that states them. Anal Bioch article only presents first initials.--> 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.{{primary source inline|date=October 2025}}<!--See note in all caps above.--><ref>{{cite journal | author = Uhlen, M. | date = 31 August 1989 | title = Magnetic Separation of DNA | journal = [[Nature (journal)|Nature (London)]] | volume = 340 | issue = 6236| pages = 733–734 | issn = | url = https://www.nature.com/articles/340733a0#citeas | access-date = 28 October 2025 | url-access = subscription | doi = 10.1038/340733a0 | pmid = 2770876 | bibcode = 1989Natur.340..733U }}{{primary source inline|date=October 2025}}</ref><ref>{{cite journal | author = Nyrén, Pål & Lundin, Arne | date = December 1985 | title = Enzymatic Method for Continuous Monitoring of Inorganic Pyrophosphate Synthesis | journal = Analytical Biochemistry | volume = 151 | issue = 2 | pages = 504–509 | issn = 0003-2697 | url = https://dx.doi.org/10.1016/0003-2697%2885%2990211-8 | access-date = 28 October 2025 | url-access = subscription | doi = 10.1016/0003-2697(85)90211-8 | pmid = 3006540 }}{{primary source inline|date=October 2025}}</ref>{{what|date=October 2025}} | |||
Specifically, a solution of three [[enzyme]]s—[[DNA polymerase]], [[Sulfate adenylyltransferase|ATP sulfurylase]], and firefly [[luciferase]]—and a deoxyribo[[nucleoside 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.{{cn|date=October 2025}} 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.{{cn|date=October 2025}} 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.{{cn|date=October 2025}} | |||
A | A second solution-based method for pyrosequencing was described in 1998 by [[Mostafa Ronaghi]], [Mathias Uhlen],<ref>{{cite web | title=School of Engineering Sciences in Chemistry, Biotechnology and Health | url=https://www.kth.se/en/bio/research/proteomics/proteomics-researchers/mathias-uhlen-1.67763 }}</ref> and [[Pål Nyrén|Pål Nyren]].{{primary source inline|date=October 2025}}<!--See note in all caps above.-->In this alternative method, an additional enzyme, [[apyrase]], is introduced to remove nucleotides that are not incorporated by the DNA polymerase.{{cn|date=October 2025}} 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).{{cn|date=October 2025}} An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.{{cn|date=October 2025}} | ||
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]].{{primary source inline|date=October 2025}}<!--See note in all caps above.--><ref>{{cite journal | author = 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. | author-mask = | date = 31 July 2005 | title = Genome Sequencing in Microfabricated High-Density Picolitre Reactors | journal = [[Nature (journal)|Nature (London)]] | volume = 437 | issue = 7057| pages = 376–380 | issn = | doi = 10.1038/nature03959 | pmid = 16056220| pmc = 1464427| bibcode = 2005Natur.437..376M}}{{primary source inline|date=October 2025}}</ref><ref name=":1" /> 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 [[microarray]]s.{{cn|date=October 2025}} This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.{{cn|date=October 2025}} This first next generation sequencing instrument initiated a new era in [[genomics]] research,{{says who|date=October 2025}} and to rapidly falling prices for [[DNA sequencing]],{{says who|date=October 2025}} allowing affordable [[whole genome sequencing]].{{cn|date=October 2025}} | |||
==Procedure == | ==Procedure == | ||
{{verify sources|section|date=October 2025}} | |||
[[File:How Pyrosequencing Works.svg|alt=How Pyrosequencing Works|thumb|679x679px|The chart shows how pyrosequencing works.]] | [[File:How Pyrosequencing Works.svg|alt=How Pyrosequencing Works|thumb|679x679px|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 [[chemiluminescence|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. | "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 [[chemiluminescence|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.<ref name=Qiagen>{{cite web| author = Qiagen Staff | date = 4 August 2017 | title=Pyrosequencing Technology and Platform Overview | work = Qiagen Knowledge & Support/Knowledge Hub/Technology & Research/Pyrosequencing Resource Center (Qiagen.com) | url=https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/technology-overview/| access-date=4 August 2017}}{{better source|date=October 2025}}</ref><ref name=":0" /><ref name=":1" />{{better source|date=October 2025}}<!--This commercial source will lean in the direction of the product they are selling, and so is a poorer source than a published review or other secondary source.-->{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | ||
For the solution-based version of pyrosequencing, the single-strand DNA ([[ssDNA#ssDNA|ssDNA]]) template is hybridized to a sequencing [[Primer (molecular biology)|primer]] and incubated with the enzymes [[DNA polymerase]], [[ATP sulfurylase]], [[luciferase]] and [[apyrase]], and with the substrates [[adenosine 5´ phosphosulfate]] (APS) and [[luciferin]]. | For the solution-based version of pyrosequencing, the single-strand DNA ([[ssDNA#ssDNA|ssDNA]]) template is hybridized to a sequencing [[Primer (molecular biology)|primer]] and incubated with the enzymes [[DNA polymerase]], [[ATP sulfurylase]], [[luciferase]] and [[apyrase]], and with the substrates [[adenosine 5´ phosphosulfate]] (APS) and [[luciferin]].<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | ||
# The addition of one of the four [[deoxynucleotide triphosphate]]s | |||
# ATP sulfurylase converts PPi to [[Adenosine triphosphate|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. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program. | # The addition of one of the four [[deoxynucleotide triphosphate]]s initiates the second step; [[dNTP]]s)—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).<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | ||
# Unincorporated nucleotides and ATP are degraded by the [[apyrase]], and the reaction can restart with another nucleotide. | |||
# ATP sulfurylase converts PPi to [[Adenosine triphosphate|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.{{what|date=October 2025}} The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | |||
# Unincorporated nucleotides and ATP are degraded by the [[apyrase]], and the reaction can restart with another nucleotide.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | |||
The process can be represented by the following equations: | The process can be represented by the following equations: | ||
* PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase); | * PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase); | ||
* ATP + luciferin + O2 → AMP + PPi + oxyluciferin + {{CO2}} + hv (catalyzed by luciferase); | * ATP + luciferin + O2 → AMP + PPi + oxyluciferin + {{CO2}} + hv (catalyzed by luciferase); | ||
where | where PPi is pyrophosphate, APS is adenosine 5-phosphosulfate, ATP is adenosine triphosphate, O2 is di[[oxygen]], AMP is adenosine monophosphate, {{CO2}} is carbon dioxide, and hv is light.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.--> | ||
==Limitations== | ==Limitations== | ||
{{unreferenced section|date=October 2025}} | |||
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 [[DNA sequencing#Chain-termination methods|chain termination]] methods (e.g. Sanger sequencing).{{cn|date=October 2025}} This can make the process of [[genome assembly]] more difficult, particularly for sequences containing a large amount of [[repetitive DNA]].{{cn|date=October 2025}} Also, lack of a proof-reading activity{{what|date=October 2025}} limits accuracy of this method.{{cn|date=October 2025}} | |||
==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.<ref name=BiotageHistory/>{{verification needed|date=October 2025}} Pyrosequencing AB was listed on the [[Stockholm Stock Exchange]] in 1999.<ref name=BiotageHistory/>{{verification needed|date=October 2025}} When Pyrosequencing AB acquired Biotage LLC, a U.S.-based company, and other companies, in 2003, the company was renamed Biotage AB.<ref name=BiotageHistory>{{Cite web |author=Biotage Staff |title=Biotage History |url=https://www.biotage.com/biotage-history |access-date=2022-09-19 |work=Biotage.com |language=en}}</ref> The pyrosequencing and other biomedical units of Biotage AB were sold to [[Qiagen]] in 2008.<ref name=BiotageHistory/>{{verification needed|date=October 2025}} The pyrosequencing technology was licensed to [[454 Life Sciences]].{{when|date=October 2025}}{{cn|date=October 2025}} 454 developed an array-based pyrosequencing technology that emerged as a platform for [[DNA sequencing#High-throughput sequencing|large-scale DNA sequencing]], including [[genome project|genome sequencing]] and [[metagenomics]].{{cn|date=October 2025}} | |||
== | [[Hoffmann-La Roche|Roche]] acquired [[454 Life Sciences]],{{when|date=October 2025}}{{cn|date=October 2025}} and announced the discontinuation of the 454 sequencing platform in 2013.<ref>{{cite news|author =Hollmer, Mark| date=October 17, 2013 | title=Roche to Close 454 Life Sciences as it Reduces Gene Sequencing Focus | work = [[Fierce Biotech]] | url=http://www.fiercebiotech.com/medical-devices/roche-to-close-454-life-sciences-as-it-reduces-gene-sequencing-focus| access-date = 28 October 2025}}</ref> The 454 sequencing platform was replaced, in part, by [[Illumina dye sequencing]], and by Applied Biosystems sequencing products.{{cn|date=October 2025}} | ||
==Further reading== | |||
* {{cite journal| author= Harrington, C.T.; Lin, E.I.; Olson, M.T. & Eshleman, J.R. | date = September 1, 2013 | title = Fundamentals of Pyrosequencing | journal = Arch. Pathol. Lab. Med. | volume = 137 | issue = 9 | pages = 1296–1303 | url = https://meridian.allenpress.com/aplm/article/137/9/1296/193658/Fundamentals-of-Pyrosequencing | url-access =subscription | access-date = 29 October 2025 | doi = 10.5858/arpa.2012-0463-RA | pmid = 23991743 }} For the corresponding PDF document, see [https://allen.silverchair-cdn.com/allen/content_public/journal/aplm/137/9/10.5858_arpa.2012-0463-ra/2/arpa_2012-0463-ra.pdf?Expires=1764740125&Signature=G9NXpBjO~irAudtDoIXycecX~4lHevw~xMEYSc~6DZTDT~ctj6sICV4afDIAu6Nkza6Hfky5~5hE3hTuYpnUenRfUj2VLfIz5BQx17FoIp3sI4CoxjzI16ACtBq~cLi4A48-CAuPrpsJY5-4bWolqNHQpc5GCTdzLl1VkYknDcc4TlRYBt89l7XmfJ-bFIJak0S2T8M7L-Dth19D0z8UPIPbIo4nuaPergMq2Gf0q0Rn9GZlLLaeePPuiEgcAISUYX3PezwNrAUGXmeEnuBSAmUAhlH82h0zNcibUsdlUpSqpKQRjMg5beyISWDK8zsZ~xrSMgjraiZ9BxZZHLNkNA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA this link]. | |||
* {{cite journal|author=Metzker M. |title=Emerging Technologies in DNA Sequencing|journal=Genome Research |date=2005| pmid=16339375|volume=15|pages=1767–76|doi=10.1101/gr.3770505|issue=12|doi-access=free}} | |||
==References== | ==References== | ||
{{Reflist}} | {{Reflist}} | ||
[[Category:Biotechnology]] | [[Category:Biotechnology]] | ||
Latest revision as of 19:45, 31 December 2025
Template:Short description Template:Use dmy dates Pyrosequencing is a non-electrophoretic DNA sequencing (determining the order of nucleotides in DNA) method 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
Script error: No such module "Unsubst". The principle of pyrosequencing was first described in 1993 by P. Nyrén, B. Pettersson, and M. Uhlen.Template:Primary source inline[1][2][3] 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.Template:Primary source inline[4][5]Script error: No such module "Unsubst".
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.Script error: No such module "Unsubst". 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.Script error: No such module "Unsubst". 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.Script error: No such module "Unsubst".
A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi, [Mathias Uhlen],[6] and Pål Nyren.Template:Primary source inlineIn this alternative method, an additional enzyme, apyrase, is introduced to remove nucleotides that are not incorporated by the DNA polymerase.Script error: No such module "Unsubst". 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).Script error: No such module "Unsubst". An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.Script error: No such module "Unsubst".
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.Template:Primary source inline[7][3] 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.Script error: No such module "Unsubst". This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.Script error: No such module "Unsubst". This first next generation sequencing instrument initiated a new era in genomics research,
- REDIRECT Template:According to whom and to rapidly falling prices for DNA sequencing,
- REDIRECT Template:According to whom allowing affordable whole genome sequencing.Script error: No such module "Unsubst".
Procedure
"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.[8][1][3]Template:Better sourceScript error: No such module "Unsubst".
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.[8]Script error: No such module "Unsubst".
- 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).[8]Script error: No such module "Unsubst".
- 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.Script error: No such module "Unsubst". The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.[8]Script error: No such module "Unsubst".
- Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.[8]Script error: No such module "Unsubst".
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.[8]Script error: No such module "Unsubst".
Limitations
Script error: No such module "Unsubst". 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).Script error: No such module "Unsubst". This can make the process of genome assembly more difficult, particularly for sequences containing a large amount of repetitive DNA.Script error: No such module "Unsubst". Also, lack of a proof-reading activityScript error: No such module "Unsubst". limits accuracy of this method.Script error: No such module "Unsubst".
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.[9]Script error: No such module "Unsubst". Pyrosequencing AB was listed on the Stockholm Stock Exchange in 1999.[9]Script error: No such module "Unsubst". When Pyrosequencing AB acquired Biotage LLC, a U.S.-based company, and other companies, in 2003, the company was renamed Biotage AB.[9] The pyrosequencing and other biomedical units of Biotage AB were sold to Qiagen in 2008.[9]Script error: No such module "Unsubst". The pyrosequencing technology was licensed to 454 Life Sciences.Script error: No such module "Unsubst".Script error: No such module "Unsubst". 454 developed an array-based pyrosequencing technology that emerged as a platform for large-scale DNA sequencing, including genome sequencing and metagenomics.Script error: No such module "Unsubst".
Roche acquired 454 Life Sciences,Script error: No such module "Unsubst".Script error: No such module "Unsubst". and announced the discontinuation of the 454 sequencing platform in 2013.[10] The 454 sequencing platform was replaced, in part, by Illumina dye sequencing, and by Applied Biosystems sequencing products.Script error: No such module "Unsubst".
Further reading
- Script error: No such module "Citation/CS1". For the corresponding PDF document, see this link.
- Script error: No such module "Citation/CS1".
References
<templatestyles src="Reflist/styles.css" />
- ↑ a b Script error: No such module "Citation/CS1".
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