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 phase 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. A third microfluidic variant of the pyrosequencing method was described in 2005 by Jonathan Rothberg and co-workers at the company 454 Life Sciences.

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related publications (16)

Related people (7)

Related units (4)

Related concepts (5)

Related lectures (2)

Related MOOCs (6)

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, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, DNA Genographic Projects and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics.

Sequence assembly

In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically, the short fragments (reads) result from shotgun sequencing genomic DNA, or gene transcript (ESTs).

Whole genome sequencing

Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a single time. This entails sequencing all of an organism's chromosomal DNA as well as DNA contained in the mitochondria and, for plants, in the chloroplast. Whole genome sequencing has largely been used as a research tool, but was being introduced to clinics in 2014.

Comparison of electrical and optical transduction modes of DNA-wrapped SWCNT nanosensors for the reversible detection of neurotransmitters.

Jürgen Brugger, Giovanni Boero, Nergiz Sahin Solmaz

In this study, we compare the electrical and optical signal transduction of nanoscale biosensors based on single -walled carbon nanotubes (SWCNTs). Solution processable single-stranded (ss) DNA-wrapped SWCNTs were used for the fabrication of the distinct s ...

ELSEVIER ADVANCED TECHNOLOGY2022

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies

Vytautas Navikas

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barrie ...

AMER CHEMICAL SOC2019

Genetic diversity of two Daphnia-infecting microsporidian parasites, based on sequence variation in the internal transcribed spacer region

Stefan Jean Laurent

Background: Microsporidia are spore-forming obligate intracellular parasites that include both emerging pathogens and economically important disease agents. However, little is known about the genetic diversity of microsporidia. Here, we investigated patter ...

Biomed Central Ltd2016

This course will provide the fundamental knowledge in neuroscience required to understand how the brain is organised and how function at multiple scales is integrated to give rise to cognition and beh

Covers short-read sequencing techniques using fluorescent dye and pyrosequencing.

Explores DNA sequencing methods like Sanger, pyrosequencing, and synthesis technologies.