Pyrosequencing is a DNA sequencing method that detects nucleotide incorporation through light emission during a 'sequencing by synthesis' process. Developed from principles established in the early 1990s, it has evolved through various methods, including solid-phase and microfluidic approaches, facilitating high-throughput sequencing. Despite its advantages, pyrosequencing faces limitations in read length and accuracy due to the absence of proofreading activity.

1. Pyrosequencing
Dr. Naveen Gaurav
Associate Professor and Head
Department of Biotechnology
Shri Guru Ram Rai University
Dehradun

2. Pyrosequencing
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in
DNA) 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 pyrosequencing.
The principle of Pyrosequencing was first described in 1993 by Bertil Pettersson, Mathias
Uhlen and Pål Nyren by combining the solid phas sequencing method using streptavidin coated
magnetic beads with recombinant DNA polymerase lacking 3´to 5´exonuclease activity (proof-
reading) and luminescence detection using the firefly luciferase enzyme. A mixture of
three enzymes (DNA polymerase, ATP sulfurylase and firefly luciferase) and a nucleotide (dNTP)
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.
A second solution-based method for Pyrosequencing was described in 1998 by Mostafa
Ronaghi, Mathias Uhlen and Pål Nyren. In this alternative method, an additional
enzyme apyrase is introduced to remove nucleotides that are not incorporated by the DNA
polymerase. This enabled the enzyme mixture including the DNA polymerase, the luciferase and
the apyrase 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.

3. A third microfluidic variant of the Pyrosequencing method was described in
2005 by Jonathan Rothberg and co-workers at the company 454 Life Sciences. 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 microfabricated microarray. 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 genomics research, with rapidly falling prices for DNA sequencing allowing whole genome
sequencing at affordable prices.
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.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.

4. The addition of one of the four deoxynucleotide triphosphates (dNTPs) (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 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.

6. Limitations
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). This can make the
process of genome assembly more difficult, particularly for sequences containing a large
amount of repetitive DNA. Lack of proof-reading activity limits accuracy of this method.
Commercialization
The company Pyrosequencing AB 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. Pyrosequencing
AB was listed on the Stockholm Stock Exchange in 1999. It was renamed to Biotage in
2003. The pyrosequencing business line was acquired by Qiagen in 2008. Pyrosequencing
technology was further licensed to 454 Life Sciences. 454 developed an array-based
pyrosequencing technology which emerged as a platform for large-scale DNA sequencing,
including genome sequencing and metagenomics.
Roche announced the discontinuation of the 454 sequencing platform in 2013 when its
technology became noncompetitive.
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