Pyrosequencing

<h2 id="procedure">Procedure</h2>

<p>"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 <a href="/facts/DNA_polymerase/xtNLncBu">DNA polymerase</a> (a DNA synthesizing enzyme) with another <a href="/facts/Chemiluminescence/9LLwID4d">chemoluminescent</a> <a href="/facts/Enzyme/DSI1Fh7y">enzyme</a>.  Essentially, the method allows sequencing a single strand of <a href="/facts/DNA/nB89Iauz">DNA</a> 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 <a href="/facts/Nucleotides/pQKEwlJI">nucleotides</a> 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.<a class="footnote-ref" id="fnref:6" href="#fn:6"><sup>6</sup></a>
</p><p>For the solution-based version of pyrosequencing, the single-strand DNA (<a href="/facts/SsDNA/nB89Iauz">ssDNA</a>) template is hybridized to a sequencing <a href="/facts/Primer_(molecular_biology)/I3fBfwte">primer</a> and incubated with the enzymes <a href="/facts/DNA_polymerase/xtNLncBu">DNA polymerase</a>, <a href="/facts/ATP_sulfurylase/pBh0SfrW">ATP sulfurylase</a>, <a href="/facts/Luciferase/7yl8dytU">luciferase</a> and <a href="/facts/Apyrase/cMh8Ua7M">apyrase</a>, and with the substrates <a href="/facts/Adenosine_5%C2%B4_phosphosulfate/Xmab8Gqj">adenosine 5´ phosphosulfate</a> (APS) and <a href="/facts/Luciferin/9AzPgE9n">luciferin</a>.
</p>
<ol><li>The addition of one of the four <a href="/facts/Deoxynucleotide_triphosphate/pQKEwlJI">deoxynucleotide triphosphates</a> (<a href="/facts/DNTP/cmhLQj6s">dNTPs</a>) (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 <a href="/facts/Pyrophosphate/L9FPEkhF">pyrophosphate</a> (PPi).</li>
<li>ATP sulfurylase converts PPi to <a href="/facts/Adenosine_triphosphate/nB9bwgcU">ATP</a> 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.</li>
<li>Unincorporated nucleotides and ATP are degraded by the <a href="/facts/Apyrase/cMh8Ua7M">apyrase</a>, and the reaction can restart with another nucleotide.</li></ol>
<p>The process can be represented by the following equations:
</p>
<ul><li>PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase);</li>
<li>ATP + luciferin + O2 → AMP + PPi + oxyluciferin + CO2 + hv (catalyzed by luciferase);</li></ul>
<p>where:
</p>
<ul><li>PPi is pyrophosphate</li>
<li>APS is adenosine 5-phosphosulfate;</li>
<li>ATP is adenosine triphosphate;</li>
<li>O2 is oxygen molecule;</li>
<li>AMP is adenosine monophosphate;</li>
<li>CO2 is carbon dioxide;</li>
<li>hv is light.</li></ul>
<h2 id="limitations">Limitations</h2>
<p>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 <a href="/facts/DNA_sequencing/hsPsNCQW">chain termination</a> methods (e.g. Sanger sequencing).  This can make the process of <a href="/facts/Genome_assembly/owEm30vn">genome assembly</a> more difficult, particularly for sequences containing a large amount of <a href="/facts/Repetitive_DNA/hPosWry7">repetitive DNA</a>. Lack of proof-reading activity limits accuracy of this method.
</p>
<h2 id="commercialization">Commercialization</h2>
<p>The company Pyrosequencing AB in <a href="/facts/Uppsala,_Sweden/X3HzSeYN">Uppsala, Sweden</a> was founded with <a href="/facts/Venture_capital/yeH3m7zo">venture capital</a> provided by <a href="/facts/HealthCap/6S0Ff80L">HealthCap</a> in order to commercialize machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique. Pyrosequencing AB was listed on the <a href="/facts/Stockholm_Stock_Exchange/wAVPR550">Stockholm Stock Exchange</a> in 1999. It was renamed to Biotage in 2003.<a class="footnote-ref" id="fnref:7" href="#fn:7"><sup>7</sup></a> The pyrosequencing business line was acquired by <a href="/facts/Qiagen/zpepVIfc">Qiagen</a> in 2008.  Pyrosequencing technology was further licensed to <a href="/facts/454_Life_Sciences/uJckH5GR">454 Life Sciences</a>. 454 developed an array-based pyrosequencing technology which emerged as a platform for <a href="/facts/DNA_sequencing/hsPsNCQW">large-scale DNA sequencing</a>, including <a href="/facts/Genome_project/sIYftGNF">genome sequencing</a> and <a href="/facts/Metagenomics/GWFeMd7z">metagenomics</a>.
</p><p><a href="/facts/Hoffmann-La_Roche/e4Wdasmz">Roche</a> announced the discontinuation of the 454 sequencing platform in 2013.<a class="footnote-ref" id="fnref:8" href="#fn:8"><sup>8</sup></a>
</p>

<h3>Further reading</h3>
<ul><li>Metzker M. (2005). <a href="https://doi.org/10.1101%2Fgr.3770505">"Emerging Technologies in DNA Sequencing"</a>. <i>Genome Research</i>. 15 (12): 1767–76. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1101%2Fgr.3770505">10.1101/gr.3770505</a>. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/16339375">16339375</a>.</li></ul>

<h2 id="references">References</h2>

<ol>
<li id="fn:1"><p>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 <a href="https://doi.org/10.1006/abio.1993.1024" target="_blank">https://doi.org/10.1006/abio.1993.1024</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li>
<li id="fn:2"><p>Uhlen (1989) ”Magnetic separation of DNA”  Nature 340: 733-4, https://doi.org/10.1038/340733a0 <a href="https://doi.org/10.1038/340733a0" target="_blank">https://doi.org/10.1038/340733a0</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li>
<li id="fn:3"><p>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 <a href="https://doi.org/10.1016/0003-2697(85)90211-8" target="_blank">https://doi.org/10.1016/0003-2697(85)90211-8</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li>
<li id="fn:4"><p>Ronaghi, Mostafa; Uhlén, Mathias; Nyrén, Pål (1998-07-17). "A Sequencing Method Based on Real-Time Pyrophosphate". Science. 281 (5375): 363–365. doi:10.1126/science.281.5375.363. PMID 9705713. S2CID 26331871. <a href="https://www.science.org/doi/abs/10.1126/science.281.5375.363" target="_blank">https://www.science.org/doi/abs/10.1126/science.281.5375.363</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li>
<li id="fn:5"><p>Marguiles et al (2005) “Genome sequencing in microfabricated high-density picolitre reactors” Nature 437, 376-380.  https://doi.org/doi:10.1038/nature03959; <a href="https://doi.org/doi:10.1038/nature03959" target="_blank">https://doi.org/doi:10.1038/nature03959</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li>
<li id="fn:6"><p>QIAGEN. "Pyrosequencing Technology and Platform Overview". Retrieved 4 August 2017. <a href="https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/technology-overview/" target="_blank">https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/technology-overview/</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li>
<li id="fn:7"><p>Biotage. "Biotage History". www.biotage.com. Retrieved 2022-09-19. <a href="https://www.biotage.com/biotage-history" target="_blank">https://www.biotage.com/biotage-history</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li>
<li id="fn:8"><p>Hollmer, Mark (October 17, 2013). "Roche to close 454 Life Sciences as it reduces gene sequencing focus". Fierce Biotech. <a href="http://www.fiercebiotech.com/medical-devices/roche-to-close-454-life-sciences-as-it-reduces-gene-sequencing-focus" target="_blank">http://www.fiercebiotech.com/medical-devices/roche-to-close-454-life-sciences-as-it-reduces-gene-sequencing-focus</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li>
</ol>

Pyrosequencing open-in-new

Pyrosequencing