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Pyrosequencing: Principle, Steps, Reactions, Types, Agricultural Microbiology (27)

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Pyrosequencing is a sequencing method that determines the sequence of
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nucleotides by detecting the release of pyrophosphate (PPi) during nucleotide
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incorporation and monitors DNA synthesis in real-time.
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Pyrosequencing relies on light detection which occurs due to a chain reaction
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initiated by the release of pyrophosphate. Unlike Sanger sequencing, this method
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does not use fluorescently labeled nucleotides and does not rely on
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electrophoresis for visualization. Pyrosequencing is useful for applications that
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require short reads and real-time data analysis. This method has now been largely
replaced by several new technologies but it laid the groundwork for the Microscopy (30)

development of next-generation sequencing (NGS) technologies. Molecular Biology (93)

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Pyrosequencing

Table of Contents
Historical Background
Principle of Pyrosequencing
Process/Steps of Pyrosequencing
1. Sample Preparation
Principle of Pyrosequencing
The principle of pyrosequencing is based on the sequencing-by-synthesis method.
It is based on the addition of nucleotides to a single-stranded DNA template and
the detection of the release of pyrophosphate as each nucleotide is added. The
released PPi initiates a series of reactions that produce a light signal which is
used to determine the DNA sequence.

In the pyrosequencing process, DNA polymerase adds nucleotides sequentially to Post-Fertilization in Plants: Seed
the template strand and releases PPi when the nucleotide matches the and Fruit Development
complementary base. ATP sulfurylase converts the released PPi into adenosine Mitochondrial DNA analysis as a
triphosphate (ATP). Then the enzyme luciferase uses the ATP to convert the Forensic Tool
substrate luciferin into oxyluciferin which emits light as a result. The light emitted Standard Precautions (Infection
is detected by a sensor and recorded as peaks called Pyrogram. The intensity of Control) in Healthcare
the emitted light correlates with the amount of PPi released and thus reflects the Long-Read Sequencing: Principle,
number of nucleotides added. Any unincorporated or excess nucleotides are Types, Process, Uses
broken down by the enzyme apyrase before the next nucleotide is added.
Pollen-Pistil Interaction: Key
Processes and Significance

Principle of Pyrosequencing. Image Source: QIAGEN.

Process/Steps of Pyrosequencing
1. Sample Preparation
DNA of interest is extracted from biological samples using different suitable
methods like mechanical disruption and chemical lysis.
The extracted DNA is fragmented into small pieces with restriction enzymes or
by using mechanical methods.

2. PCR Amplification
The DNA template for sequencing is prepared by amplifying the region of
interest using PCR which generates multiple copies of each fragment.
The fragmented DNA is amplified using a biotinylated primer which labels one
strand with biotin.
The PCR product contains one biotinylated strand that is used as the template
for the sequencing reaction.
This biotin-tagged single-stranded DNA is isolated from the PCR product using
streptavidin-coated beads that bind to the biotin label.
The template DNA is hybridized with a sequencing primer and then added to the
pyrosequencing reaction.

3. Sequencing Reaction
The necessary reagents including the template DNA, enzymes, and substrates
are loaded into the sequencing instrument to initiate the sequencing reaction.
The sequencing reaction begins with the addition of nucleotides to the template
DNA.
DNA polymerase adds the complementary nucleotide into the DNA strand. This
releases pyrophosphate which initiates an enzymatic reaction cascade.
DNA template + dNTP (complementary) → DNA product + PPi + H+

The released PPi is converted into ATP by the enzyme ATP sulfurylase in the
presence of the substrate adenosine 5′ phosphosulfate (APS).
PPi + APS → ATP + SO42−

The ATP generated is used to convert the luciferin substrate to oxyluciferin by

the enzyme luciferase. This produces light signals that indicate how many
nucleotides were added.
ATP + luciferin + O2 → AMP + PPi + oxyluciferin + CO2 ​+ light

The enzyme apyrase degrades ATP and any unused or unincorporated

nucleotides.
Unincorporated nucleotide + H2O → Nucleoside + Pi
 The intensity of the emitted light is detected with sensors like a charge-coupled
device (CCD) camera and recorded as peaks.

4. Sequence Analysis
The sequencing reaction generates pyrograms which is a graphical
representation of the light signals. It displays the sequence of light peaks
corresponding to the sequence of nucleotides added.
The light signals are analyzed to determine the sequence of nucleotides.
Multiple fragments are assembled into a complete DNA sequence using
bioinformatics tools.

Pyrosequencing Video Animation

Types of Pyrosequencing
Advantages of Pyrosequencing
Pyrosequencing provides real-time sequencing data that allows monitoring of
the sequencing process as it occurs.
It is a high-throughput method that can process multiple samples
simultaneously which is useful in large-scale genome sequencing.
Pyrosequencing results are directly converted into Pyrogram peaks. This
removes the need for post-sequencing electrophoresis for analysis which
reduces time and cost.
Pyrosequencing involves fewer sample preparation steps and pre-processing
steps compared to other sequencing methods.
Pyrosequencing does not require fluorolabeling of nucleotides. The use of
natural nucleotides reduces costs and simplifies the process.

Limitations of Pyrosequencing
Pyrosequencing produces shorter read lengths compared to other sequencing
methods. This can limit its use for sequencing large genomes and repetitive
sequences.
Pyrosequencing cannot accurately detect long homopolymers or sequences
that contain repeated nucleotides which leads to sequencing errors.
Although pyrosequencing is less expensive than some next-generation
sequencing methods, the cost per base pair is higher compared to other high-
throughput methods.
Pyrosequencing has been largely replaced by more advanced sequencing
technologies that offer longer read lengths and higher throughput at a lower
cost.