Pyrosequencing

From Wikipedia, the free encyclopedia

The introduction to this article provides insufficient context for those unfamiliar with the subject matter.
Please help Wikipedia by improving the introduction according to the guidelines laid out at Wikipedia:Guide to layout. You can discuss the issue on the talk page.

Pyrosequencing is a novel method of DNA sequencing based on the "sequencing by synthesis" principle developed initially by Mostafa Ronaghi and co-workers in 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

A PCR-amplified 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.
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.
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 reaction is detected by a charge coupled device (CCD) camera and this can be analyzed in a program. Each light signal is proportional to the number of nucleotides incorporated.
Nucleotide degradation by apyrase removes dNTPs from the solution.
New nucleotides can be added and a new cycle can start.

[edit] Recent advances

The technique has been further developed by the company 454 into a technology known as 454 Sequencing. To date this is the fastest sequencing method. However, a serious limitiation 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 much more complicated, particularly for genomes which contain a large amount of repetitive DNA. Pyrosequencing is therefore 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)