Pyrosequencing

src: i.ytimg.com

Pyrosequencing is a DNA sequencing method (determining the nucleotide sequence in DNA) based on the principle of "sorting by synthesis", where sorting is done by detecting nucleotides inserted by DNA polymerases. Pyrosequencing relies on light detection based on chain reactions when the pyrophosphate is released.

The Pyrosequencing Principles were first described in 1993 by Bertil Pettersson, Mathias Uhlen and PÃÆ'  ¥ l Nyren by combining solid phase sequence methods using streptavidin coated magnetic beads with recombinant DNA polymerase that had no activity of 3Ã,Â'to 5Ã,Â'exonuclease ( proof-reading) and luminescence detection using the firer-liferiferase enzyme. Mixtures of three enzymes (DNA polymerase, ATP sulfurylase and firefly luciferase) and nucleotides (dNTP) are added to a single stranded DNA to be sequenced and nucleotide incorporation followed by measuring the emitted light. The intensity of light determines whether 0, 1 or more nucleotides have been entered, thus indicating how many complementary nucleotides are present in the mold 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 from a single stranded template is determined.

The second solution-based method for Pyrosequencing was described in 1998 by Mostafa Ronaghi, Mathias Uhlen and PÃÆ'nmen l Nyren. In this alternative method, an additional apyrase enzyme was introduced to remove the nucleotides not included by the DNA polymerase. This enables enzyme blends including DNA polymerases, luciferases and apyrases to be added at baseline and stored throughout the procedure, thus providing simple settings suitable for automation. Automated instruments based on this principle were introduced to the market the following year by Pyrosequencing companies.

The third microfluidic variant of the Pyrosequencing method was described in 2005 by Jonathan Rothberg and co-workers at the 454 Life Sciences company. An alternative approach to Pyrosequencing is based on the original principle of attaching DNA to be sorted into solid support and they show that sorting can be done in a very parallel way using microfabricated microarray. This allows sequencing of DNA with high throughput and automatic instruments introduced into the market. It became the next generation of sequencing instruments that started a new era in genomic research, with rapid price reductions for DNA sequencing that allowed the entire sequencing of genomes at affordable prices.

Video Pyrosequencing

Procedure

"Sequencing by synthesis" involves taking a single strand of DNA to be sorted and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (DNA synthesis enzyme) with other chemoluminescent enzymes. Basically, this method allows the sequencing of a single strand of DNA by synthesizing a complementary strand along it, one base pair at a time, and detecting a base actually added at each step. The template DNA is immobile, and the Nucleotide A, C, G, and T solutions are sequentially added and removed from the reaction. Light is generated only when the nucleotide solution completes the first base of the unpaired template. The order of solutions that produce chemiluminescent signals allows for the determination of the order of templates.

For the solution-based version of Pyrosequencing, single-strand DNA template (ssDNA) in hybridization became sequencing primers and incubated with DNA polymerase enzymes, ATP sulfurylase, luciferase and apyrase, and with adenosine 5α phosphosulfate substrates (APS) and luciferin.

1. The addition of one of the four deoxynucleotide triphosphates (dNTPs) (dATP? s, which is not a substrate for luciferase, added in place of dATP to avoid sound) initiates the second step. DNA polymerase incorporates the correct and complementary dNTP into the template. This merger releases pyrophosphate (PPi).
2. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5Ã,Â'phosphosulfate. This ATP acts as a substrate for the conversion of luciferin-mediated luciferin to oxyluciferin which produces visible light in an amount equivalent to the amount of ATP. The light generated in the catalysis-luciferase reaction is detected by the camera and analyzed in the pyrogram.
3. Unrelated nucleotides and ATPs are subtracted by apyrases, and their reactions can be restarted with other nucleotides.

Currently, the limitation of the method is that the individual reading length of the DNA sequence is around 300-500 nucleotides, shorter than 800-1000 which can be obtained by chain termination method (eg Sanger sequence). This can make the assembly process of the genome more difficult, especially for circuits that contain large amounts of repetitive DNA.

Maps Pyrosequencing

Commercialization

The Pyrosequencing AB company in Uppsala, Sweden was established with venture capital provided by HealthCap to commercialize machines and reagents for short stretch DNA sequencing using pyrosequencing techniques. Pyrosequencing AB was listed on the Stockholm Stock Exchange in 1999. It was renamed Biotage in 2003. The pyrosequencing business line was acquired by Qiagen in 2008. Pyrosequencing technology was further licensed to 454 Life Sciences. 454 developed array-based pyrosequencing technology that has emerged as a platform for large-scale DNA sequencing. Most important is the application for sequencing and metagenomic genomes.

Roche announced the discontinuation of the 454 sequencing platform in 2013 when the technology became uncompetitive.

src: www.pnas.org

References

Further reading

• Metzker M. (2005). "Emerging Technologies in DNA Sequencing". Genome Research . 15 (12): 1767-76. doi: 10.1101/gr.3770505. PMIDÃ, 33 16339375.

Source of the article : Wikipedia