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Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle developed by Mostafa Ronaghi and Pål Nyrén (published in Analytical Biochemistry 1996 and Science 1998). For the history of the Pyrosequencing method see Methods Mol Biol. 2007. Pyrosequencing AB was started to commercialize the machine and reagent for sequencing of short stretches of DNA. Pyrosequencing AB was renamed to Biotage in 2003. Pyrosequencing technology was further licensed to 454 Life Sciences. 454 developed an array-based Pyrosequencing which has emerged as a rapid platform for large-scale DNA sequencing. Most notable are the applications for genome sequencing and metagenomics. GS FLX, the latest pyrosequencing platform by 454 Life Sciences (owned by Roche), can generate 100 million nucleotide data in a 7 hour run with a single machine. It is anticipated that the throughput would increase by 5-10 fold with the next release. Each run would cost about 8,000-9,000 USD, pushing de novo sequencing of mammalian genomes into the million dollar range.
Additional recommended knowledge
The method is based on a chemical light-producing enzymatic reaction, which is triggered when a molecular recognition event occurs. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it. Each time a nucleotide, A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which causes a light signal.
ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.
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 methods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequence containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available.
The templates for pyrosequencing can be made both by solid phase template preparation (Streptavidin coated magnetic beads) and enzymatic template preparation (Apyrase+Exonuclease).
The technique has been commercialized and further developed by 454 Life Sciences Corporation in its GS 20 and GS FLX line of sequencing machines.
Use in Research
External links and references
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Pyrosequencing". A list of authors is available in Wikipedia.|