Pyrosequencing is a method of
DNA sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, thymine, cytosine, and guanine. The ...
(determining the order of
nucleotides
Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...
in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
. Pyrosequencing relies on light detection based on a chain reaction when
pyrophosphate
In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...
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 phase sequencing method using
streptavidin
Streptavidin is a 52 Atomic mass unit, kDa protein (tetramer) purified from the bacterium ''Streptomyces avidinii''. Streptavidin Homotetramer, homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin ...
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
enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
s (
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
,
ATP sulfurylase
In enzymology, a sulfate adenylyltransferase () is an enzyme that catalyzes the chemical reaction
: ATP + sulfate ⇌ pyrophosphate + adenylyl sulfate
Thus, the two substrates of this enzyme are ATP and sulfate, whereas its two products are p ...
and firefly
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''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 RonaghiMathias Uhlenand
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
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
, the
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''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.
A third microfluidic variant of the pyrosequencing method was described in 2005 by
Jonathan Rothberg and co-workers at the company
454 Life Sciences
454 Life Sciences was a biotechnology company based in Branford, Connecticut that specialized in high-throughput DNA sequencing. It was acquired by Roche in 2007 and shut down by Roche in 2013 when its technology became noncompetitive, although ...
. 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
A microarray is a multiplex (assay), multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of biological interactions. It is a two-dimensional array on a Substrate (materials science), solid substrate—usu ...
. 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
Genomics is an interdisciplinary field of molecular biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, ...
research, with rapidly falling prices for
DNA sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, thymine, cytosine, and guanine. The ...
allowing
whole genome sequencing
Whole genome sequencing (WGS), also known as full genome sequencing or just genome sequencing, is the process of determining the entirety of the DNA sequence of an organism's genome at a single time. This entails sequencing all of an organism's ...
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 polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
(a DNA synthesizing enzyme) with another
chemoluminescent enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
. Essentially, the method allows sequencing a single strand of
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
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
Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...
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
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
) template is hybridized to a sequencing
primer and incubated with the enzymes
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
,
ATP sulfurylase
In enzymology, a sulfate adenylyltransferase () is an enzyme that catalyzes the chemical reaction
: ATP + sulfate ⇌ pyrophosphate + adenylyl sulfate
Thus, the two substrates of this enzyme are ATP and sulfate, whereas its two products are p ...
,
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'' ...
and
apyrase, and with the substrates
adenosine 5´ phosphosulfate (APS) and
luciferin
Luciferin () is a generic term for the light-emitting chemical compound, compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalyzed reaction with Oxygen, molecular oxygen. The resulting transforma ...
.
# 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
In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...
(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 + + 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;
* is carbon dioxide;
* hv is light.
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
In polymer chemistry, chain termination is any chemical reaction that ceases the formation of reactive intermediates in a chain propagation step in the course of a polymerization, effectively bringing it to a halt.
Mechanisms of 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
Repeated sequences (also known as repetitive elements, repeating units or repeats) are short or long patterns that occur in multiple copies throughout the genome. In many organisms, a significant fraction of the genomic DNA is repetitive, with ov ...
. Lack of proof-reading activity limits accuracy of this method.
Commercialization
The company Pyrosequencing AB in
Uppsala, Sweden
Uppsala ( ; ; archaically spelled ''Upsala'') is the capital of Uppsala County and the fourth-largest city in Sweden, after Stockholm, Gothenburg, and Malmö. It had 177,074 inhabitants in 2019.
Located north of the capital Stockholm, it is ...
was founded with
venture capital
Venture capital (VC) is a form of private equity financing provided by firms or funds to start-up company, startup, early-stage, and emerging companies, that have been deemed to have high growth potential or that have demonstrated high growth in ...
provided by
HealthCap
HealthCap is a specialized provider of venture capital within life sciences. HealthCap invests in innovative companies with focus on therapeutics. As of 2023, HealthCap has invested in over 125 companies since inception and completed initial public ...
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
Nasdaq Stockholm, formerly known as the Stockholm Stock Exchange (), is a stock exchange located in Frihamnen, Stockholm, Sweden. Founded in 1863, it has become the primary securities exchange of the Nordic countries. As of March 2021, a total ...
in 1999. It was renamed to Biotage in 2003. The pyrosequencing business line was acquired by
Qiagen
QIAGEN N.V. is a German-founded multinational provider of sample and assay technologies for molecular diagnostics, applied testing, academic research, and pharmaceutical research. The company operates in more than 35 offices in over 25 countries ...
in 2008. Pyrosequencing technology was further licensed to
454 Life Sciences
454 Life Sciences was a biotechnology company based in Branford, Connecticut that specialized in high-throughput DNA sequencing. It was acquired by Roche in 2007 and shut down by Roche in 2013 when its technology became noncompetitive, although ...
. 454 developed an array-based pyrosequencing technology which emerged as a platform for
large-scale DNA sequencing, including
genome sequencing
Whole genome sequencing (WGS), also known as full genome sequencing or just genome sequencing, is the process of determining the entirety of the DNA sequence of an organism's genome at a single time. This entails sequencing all of an organism's ...
and
metagenomics
Metagenomics is the study of all genetics, genetic material from all organisms in a particular environment, providing insights into their composition, diversity, and functional potential. Metagenomics has allowed researchers to profile the mic ...
.
Roche
F. Hoffmann-La Roche AG, commonly known as Roche (), is a Switzerland, Swiss multinational corporation, multinational holding healthcare company that operates worldwide under two divisions: Pharmaceuticals and Diagnostics. Its holding company, ...
announced the discontinuation of the 454 sequencing platform in 2013.
References
Further reading
* {{cite journal, author=Metzker M. , title=Emerging Technologies in DNA Sequencing, journal=Genome Research , date=2005, pmid=16339375, volume=15, pages=1767–76, doi=10.1101/gr.3770505, issue=12, doi-access=free
Biotechnology
DNA sequencing methods
Life sciences industry
Molecular biology