https://librepathology.org/w/index.php?action=history&feed=atom&title=PyrosequencingPyrosequencing - Revision history2026-04-16T15:17:40ZRevision history for this page on the wikiMediaWiki 1.36.3https://librepathology.org/w/index.php?title=Pyrosequencing&diff=49186&oldid=prevJensflorian: created2018-07-09T11:24:25Z<p>created</p>
<p><b>New page</b></p><div>'''Pyrosequencing''' is component of [[molecular pathology]].<br />
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==Methodology==<br />
* ''Sequence-by-synthesis''<br />
* Relies on light detection during synthesis.<br />
Simplified workflow:<br />
* A single strand of the DNA is immobilized by beads in a chamber.<br />
* The starting complementary primer is present.<br />
* The complementary strand is produced enzymatically by DNA polymerase.<br />
* Sequential dispensation of nucleotides (one base pair is analyzed at a time).<br />
* When the correct nucleotide is added, inorganic pyrophosphate is released.<br />
* The pyrophosphate is converted into [[ATP]] which oxidizes luciferin (and light emits).<br />
* The light emission is directly proportional to the number of nucleotides added.<br />
* Light ist recorded by a scanner and displayed as a pyrogram peak.<br />
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==Dispensation==<br />
* Order of nucleotide dispensation is important:<br />
* Cyclic dispensation ("AGCT" repeated)<br />
** Detects unknown targets.<br />
** May result in mutant and wild-type allele being out of phase.<br />
* Opimized dispensation (nucleotides dispensed as expected)<br />
** Type of mutation has to be known.<br />
** "Cleaner" pyrogram (easy to interpret).<br />
** Suboptimal dispensations may not show different pyrogram between simple hotspot and complex mutations.<br />
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==Advantages==<br />
* Superior detection limit compared to Sanger sequencing<br />
* Cost-effective.<br />
* Quantitative analyis.<br />
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==Disadvantages==<br />
* Shorter read lengths (usu. 100bp, max 400bp)<br />
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==See also==<br />
*[[Molecular pathology]].<br />
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==References==<br />
{{Reflist|1}}<br />
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==External links==<br />
*[http://pyromaker.pathology.jhmi.edu/ - a tool to generate expected pyrosequencing results].<br />
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[[Category:Molecular pathology]]</div>Jensflorian