Editing Pyrosequencing (section)

==Procedure ==
[[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.  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>{{cite web|last1=QIAGEN|title=Pyrosequencing Technology and Platform Overview|url=https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/technology-overview/|access-date=4 August 2017}}</ref>

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]].
# The addition of one of the four [[deoxynucleotide triphosphate]]s ([[dNTP]]s) (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 [[pyrophosphate]] (PPi).
# 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.
# Unincorporated nucleotides and ATP are degraded by the [[apyrase]], and the reaction can restart with another nucleotide.

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 oxygen molecule;
* AMP is adenosine monophosphate;
* {{CO2}} is carbon dioxide;
* hv is light.