Pyrosequencing
Pyrosequencing - Related Videos
12:37
Pyrosequencing for Microbial Identification and Characterization
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Cited by 21 •
2013
Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.
13:06
Pyrosequencing: A Simple Method for Accurate Genotyping
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Cited by 22 •
2008
Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.
07:24
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
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Cited by 6 •
2023
Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.
12:08
Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells
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Cited by 5 •
2016
This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).
08:40
Methyl-binding DNA capture Sequencing for Patient Tissues
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Cited by 1 •
2016
Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.
FAQs
The enzymatic cascade links each incorporation event to a measurable optical output. DNA polymerase releases pyrophosphate, which participates in reactions with ATP sulfurylase, luciferase, and apyrase. This coupling lets the system follow DNA synthesis as it occurs, rather than infer the sequence only after a separate endpoint assay.
Signal intensity provides quantitative information because a run of identical complementary bases produces a stronger light response than incorporation of a single base. This makes homopolymer stretches, or consecutive matching nucleotides, distinguishable through signal magnitude. Interpreting intensity therefore helps estimate how many identical bases were added at one position.
Rather than relying on fluorescently labeled terminators, Pyrosequencing reads the chemical consequence of nucleotide incorporation: pyrophosphate release and the subsequent light-producing enzyme reactions. This distinction supports rapid, quantitative analysis while preserving a real-time readout. The comparison is especially relevant when selecting a sequencing strategy for focused questions involving variants or disease-associated biomarkers.
A basic workflow adds complementary nucleotides one at a time, allows DNA polymerase to incorporate a matching base, and records the resulting light signal before proceeding to the next addition. The sequence is reconstructed from the order of signals, while their intensities indicate repeated identical bases. Each experimental step therefore corresponds to an interpretable sequence feature.
The method supports mutation detection, genotyping, microbial identification, methylation analysis, and targeted sequencing. These applications allow investigators to examine genetic variation, distinguish biological samples through sequence information, and assess disease-associated biomarkers. Its usefulness extends across studies that require focused sequence analysis rather than a general description of DNA synthesis.
Because light intensity reflects the number of identical bases incorporated, the readout provides more than a simple base-order signal. That quantitative feature can strengthen targeted analyses of genetic variation and disease-associated biomarkers. In biology, it helps connect sequence measurements with mutation detection, genotyping, or methylation analysis, depending on the research question.