92:) 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.
163:
248:
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
189:
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.
123:
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.
237:) (dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases
135:. 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
170:"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
185:
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
142:. 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
310:
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
392:
Nyren, Pettersson and Uhlen (1993) “Solid Phase DNA Minisequencing by an
Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay” Analytical Biochemistry 208 (1), 171-175,
346:
in order to commercialize machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique. Pyrosequencing AB was listed on the
416:
Nyren and Lundin (1985) “Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis” Analytiocal
Biochemistry 151 (2): 504-509.
537:
68:
coated magnetic beads with recombinant DNA polymerase lacking 3´to 5´exonuclease activity (proof-reading) and luminescence detection using the
41:
in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a
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is introduced to remove nucleotides that are not incorporated by the DNA polymerase. This enabled the enzyme mixture including the
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Marguiles et al (2005) “Genome sequencing in microfabricated high-density picolitre reactors” Nature 437, 376-380.
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in 1999. It was renamed to
Biotage in 2003. The pyrosequencing business line was acquired by
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358:. 454 developed an array-based pyrosequencing technology which emerged as a platform for
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A third microfluidic variant of the pyrosequencing method was described in 2005 by
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The principle of pyrosequencing was first described in 1993 by, Bertil
Pettersson,
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38:
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A second solution-based method for pyrosequencing was described in 1998 by
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45:. Pyrosequencing relies on light detection based on a chain reaction when
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For the solution-based version of pyrosequencing, the single-strand DNA (
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more difficult, particularly for sequences containing a large amount of
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17:
538:"Roche to close 454 Life Sciences as it reduces gene sequencing focus"
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announced the discontinuation of the 454 sequencing platform in 2013.
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322:. Lack of proof-reading activity limits accuracy of this method.
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methods (e.g. Sanger sequencing). This can make the process of
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181:. Essentially, the method allows sequencing a single strand of
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Uhlen (1989) ”Magnetic separation of DNA” Nature 340: 733-4,
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in 2008. Pyrosequencing technology was further licensed to
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Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17).
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The process can be represented by the following equations:
264:
PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase);
256:, and the reaction can restart with another nucleotide.
252:
Unincorporated nucleotides and ATP are degraded by the
431:"A Sequencing Method Based on Real-Time Pyrophosphate"
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ATP + luciferin + O2 → AMP + PPi + oxyluciferin + CO
107:. In this alternative method, an additional enzyme
491:"Pyrosequencing Technology and Platform Overview"
49:is released. Hence, the name pyrosequencing.
8:
418:https://doi.org/10.1016/0003-2697(85)90211-8
146:research, with rapidly falling prices for
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561:"Emerging Technologies in DNA Sequencing"
197:) template is hybridized to a sequencing
174:(a DNA synthesizing enzyme) with another
166:The chart shows how pyrosequencing works.
161:
477:https://doi.org/doi:10.1038/nature03959
385:
394:https://doi.org/10.1006/abio.1993.1024
7:
536:Hollmer, Mark (October 17, 2013).
282:APS is adenosine 5-phosphosulfate;
25:
406:https://doi.org/10.1038/340733a0
244:ATP sulfurylase converts PPi to
229:The addition of one of the four
291:AMP is adenosine monophosphate;
271:+ hv (catalyzed by luciferase);
201:and incubated with the enzymes
285:ATP is adenosine triphosphate;
131:and co-workers at the company
1:
231:deoxynucleotide triphosphates
447:10.1126/science.281.5375.363
72:enzyme. A mixture of three
219:adenosine 5´ phosphosulfate
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360:large-scale DNA sequencing
249:and analyzed in a program.
217:, and with the substrates
37:(determining the order of
348:Stockholm Stock Exchange
27:Method of DNA sequencing
152:whole genome sequencing
614:Life sciences industry
609:DNA sequencing methods
288:O2 is oxygen molecule;
167:
154:at affordable prices.
62:solid phase sequencing
165:
279:PPi is pyrophosphate
88:) and a nucleotide (
559:Metzker M. (2005).
578:10.1101/gr.3770505
298:is carbon dioxide;
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70:firefly luciferase
619:Molecular biology
513:"Biotage History"
441:(5375): 363–365.
364:genome sequencing
356:454 Life Sciences
338:was founded with
332:Pyrosequencing AB
326:Commercialization
312:chain termination
133:454 Life Sciences
129:Jonathan Rothberg
60:by combining the
16:(Redirected from
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571:(12): 1767–76.
565:Genome Research
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553:Further reading
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517:www.biotage.com
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340:venture capital
336:Uppsala, Sweden
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316:genome assembly
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207:ATP sulfurylase
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137:microfabricated
97:Mostafa Ronaghi
82:ATP sulfurylase
33:is a method of
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203:DNA polymerase
172:DNA polymerase
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148:DNA sequencing
113:DNA polymerase
78:DNA polymerase
43:DNA polymerase
35:DNA sequencing
31:Pyrosequencing
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101:Mathias Uhlen
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64:method using
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54:Mathias Uhlen
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47:pyrophosphate
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520:. Retrieved
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494:. Retrieved
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368:metagenomics
362:, including
342:provided by
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330:The company
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301:hv is light.
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169:
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84:and firefly
66:streptavidin
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30:
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306:Limitations
187:nucleotides
39:nucleotides
598:Categories
522:2022-09-19
380:References
221:(APS) and
211:luciferase
140:microarray
117:luciferase
86:luciferase
511:Biotage.
344:HealthCap
223:luciferin
158:Procedure
150:allowing
105:PĂĄl Nyren
58:PĂĄl Nyren
587:16339375
496:4 August
489:QIAGEN.
463:26331871
144:genomics
119:and the
455:9705713
435:Science
275:where:
254:apyrase
215:apyrase
121:apyrase
109:apyrase
74:enzymes
18:Biotage
585:
461:
453:
352:Qiagen
241:(PPi).
199:primer
179:enzyme
115:, the
459:S2CID
374:Roche
235:dNTPs
195:ssDNA
583:PMID
498:2017
451:PMID
366:and
213:and
103:and
90:dNTP
56:and
573:doi
443:doi
439:281
334:in
246:ATP
183:DNA
600::
581:.
569:15
567:.
563:.
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515:.
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437:.
433:.
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294:CO
225:.
209:,
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296:2
269:2
233:(
76:(
20:)
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
