Pyrosequencing: Principle, Methodology and Applications

Pyrosequencing is a widely used DNA sequencing technique that relies on detecting pyrophosphate (PPi) release during DNA synthesis. This method is different from the common chain termination approach as us ed in Sanger sequencing. Pyrosequencing offers real-time sequencing and is especially useful for shorter DNA fragments. It is very useful in the identification of genetic variations, mutations, and methylation patterns in the DNA. This Pyrosequencing Notes will help you to study the principle, methodology and applications of Pyrosequencing Technique. You can download the notes as PDF from the download link given below.

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Principle of Pyrosequencing

Ø  Pyrosequencing is based on the detection of the release of pyrophosphate (PPi) when a nucleotide is incorporated into a growing DNA strand during DNA synthesis.

Ø  The incorporation of a nucleotide triggers a cascade of enzymatic reactions that ultimately produce light, which is measured to indicate which nucleotide was added.

Steps involved in Pyrosequencing Process

(1). DNA Polymerization and release of PPi:

Ø  As a nucleotide complementary to the template DNA is incorporated by DNA polymerase, pyrophosphate (PPi) is released.

(2). Enzymatic Reactions:

Ø  The released PPi is converted to ATP by the enzyme ATP sulfurylase.

Ø  This ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin.

Ø  This conversion of luciferin to oxyluciferin generates visible light.

Ø  The intensity of light produced will be proportional to the amount of ATP generated in the previous reaction which in turn proportional to the amount of nucleotide added.

(3). Light Detection

Ø  The amount of light produced is directly proportional to the amount of nucleotide added, and this signal is detected by a sensor.

Ø  Each nucleotide incorporation generates a characteristic light peak.

(4). Apyrase Action:

Ø  After the signal is detected, the enzyme apyrase degrades any unincorporated nucleotides and ATP to reset the system for the next nucleotide addition.

Ø  This process is repeated to completely sequence the template strand.

Methodology of Pyrosequencing:

(a). Sample Preparation:

Ø  DNA is extracted from the sample of interest and amplified using PCR to generate sufficient template DNA.

(b). Template Immobilization:

Ø  The single-stranded DNA template is immobilized on a solid surface, such as beads or wells, to ensure proper sequencing.

(c). Sequential Nucleotide Addition:

Ø  Nucleotides (A, T, C, G) are added one at a time to the reaction mixture.

Ø  For the source of Adenine (A), instead of dATP dATPαS is added which is not a substrate for a luciferase.

Ø  If the added nucleotide is complementary to the template, DNA polymerase incorporates it, releasing PPi.

(d). Signal Detection:

Ø  The incorporation of a nucleotide is accompanied by a burst of light, whose intensity is measured to determine the sequence of the DNA.

(e). Data Analysis:

Ø  The sequence of the DNA template is inferred by analyzing the pattern and intensity of light generated as each nucleotide is incorporated.

Applications of Pyrosequencing:

(1). SNP Genotyping:

Ø  Pyrosequencing is widely used for identifying single nucleotide polymorphisms (SNPs) in various organisms, helping in disease diagnostics and population genetics studies.

(2). Methylation Analysis:

Ø  It is a powerful tool for assessing DNA methylation, an important epigenetic modification linked to gene expression regulation and disease states like cancer.

(3). Mutation Detection:

Ø  Pyrosequencing is sensitive enough to detect mutations and variations in specific genes, making it useful in clinical settings, such as cancer diagnostics and monitoring.

(4). Microbial Identification:

Ø  It can be employed for microbial typing and determining the diversity of microbial communities by analyzing 16S rRNA gene sequences.

(5). Pharmacogenomics:

Ø  The technique is used to investigate genetic variations that affect drug metabolism, aiding in personalized medicine.

Limitations of Pyrosequencing:

(i). Short Read Length:

Ø  Pyrosequencing is typically limited to sequencing short DNA fragments, generally around 100-200 base pairs, making it unsuitable for large-scale sequencing projects like whole-genome sequencing.

(ii). Errors with Homopolymer Sequences:

Ø  Pyrosequencing struggles with accurately reading homopolymeric regions (sequences with multiple identical bases in a row), which can lead to errors in the sequence interpretation.

(iii). Higher Cost:

Ø  Compared to other sequencing technologies, pyrosequencing can be more expensive due to the need for specialized equipment and reagents.

(iv). Complex Data Analysis:

Ø  The data generated from pyrosequencing requires sophisticated software for analysis, especially when dealing with complex genetic variations.

Comparison of Pyrosequencing and Sanger Sequencing

Summary: Pyrosequencing is a powerful, fast, and real-time DNA sequencing technology with significant applications in genomics, clinical diagnostics, and epigenetics. Despite its limitations, such as short read lengths and issues with homopolymers, it remains valuable for targeted sequencing tasks. Sanger sequencing, while slower, provides higher accuracy for longer DNA fragments and is still widely used for sequencing individual genes and smaller projects.

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