Pyrosequencing

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Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle developed initially by Pål Nyrén and co-workers during a period from 1985 to the late 1990s, then further by Biotage. The method is based on a chemiluminescent 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 results in a light signal. It is a method primarily used for sequencing of short stretches of DNA, SNP detection and methylation analysis. Such analyses are crucial for biological research, genetics and some medical and forensic applications. Pyrosequencing is fully automated, reliable and accurate, and large numbers of samples can be analysed in a short time. Pyrosequencing methods have been pursued to reduce costs relative to other automated sequencing methods.

[edit] Procedure

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.

  1. The addition of one of the four deoxynucleotide triphosphates (dNTPs) initiates the second step. DNA polymerase incorporate complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
  2. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed

[edit] Recent advances

The technique has been further developed by the company 454 into a technology known as 454 Pyrosequencing. To date this is the fastest sequencing method. However, a limitation of the method is that read lengths are currently somewhat shorter than those obtained by di-deoxy nucleotide based chain termination methods, which makes the process of genome assembly more complicated, particularly for genomes which contain a large amount of repetitive DNA. 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)

[edit] External links and references

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