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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 given it.
The principle of pyrosequencing was first described in 1993 by P. Nyrén, B. Pettersson, and M. Uhlen.[non-primary source needed] The technique combines solid phase sequencing, and use of streptavidin-coated magnetic beads, a recombinant DNA polymerase lacking 3´-to-5´exonuclease activity (proof-reading), and luminescence detection of inorganic pyrophosphate using the firefly luciferase enzyme.[non-primary source needed][clarification needed]
Specifically, a solution of three enzymes—DNA polymerase, ATP sulfurylase, and firefly luciferase—and a deoxyribonucleoside triphosphate (dNTP) are added to single stranded DNA to be sequenced, and the incorporation of nucleotide is followed, measuring the light emitted as a consequence of inorganic pyrophosphate production.[citation needed] 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.[citation needed] The nucleotide mixture is removed before a next nucleotide mixture is added, and the process is repeated for each of the four nucleotides, until the DNA sequence of the single stranded template is determined.[citation needed]
A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi, [Mathias Uhlen], and Pål Nyren.[non-primary source needed] In this alternative method, an additional enzyme, apyrase, is introduced to remove nucleotides that are not incorporated by the DNA polymerase.[citation needed] This enables the enzyme mixture— DNA polymerase, luciferase, and apyrase—to be added when sequencing is initiated, and kept in the reaction solution throughout the procedure (thus enabling easier automation).[citation needed] An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.[citation needed]
A third variant, a microfluidic pyrosequencing method, was described in 2005 by an industrial research team led by Jonathan Rothberg, at the company 454 Life Sciences.[non-primary source needed] This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support; Rothberg and co-workers demonstrated that sequencing could be performed in a highly parallel manner using a microfabrication and microarrays.[citation needed] This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.[citation needed] This first next generation sequencing instrument initiated a new era in genomics research,[according to whom?] and to rapidly falling prices for DNA sequencing,[according to whom?] allowing affordable whole genome sequencing.[citation needed]
"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.[better source needed][verification needed]
For the solution-based version of pyrosequencing, the single-strand DNA (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.[verification needed]
The process can be represented by the following equations:
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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 given it.
The principle of pyrosequencing was first described in 1993 by P. Nyrén, B. Pettersson, and M. Uhlen.[non-primary source needed] The technique combines solid phase sequencing, and use of streptavidin-coated magnetic beads, a recombinant DNA polymerase lacking 3´-to-5´exonuclease activity (proof-reading), and luminescence detection of inorganic pyrophosphate using the firefly luciferase enzyme.[non-primary source needed][clarification needed]
Specifically, a solution of three enzymes—DNA polymerase, ATP sulfurylase, and firefly luciferase—and a deoxyribonucleoside triphosphate (dNTP) are added to single stranded DNA to be sequenced, and the incorporation of nucleotide is followed, measuring the light emitted as a consequence of inorganic pyrophosphate production.[citation needed] 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.[citation needed] The nucleotide mixture is removed before a next nucleotide mixture is added, and the process is repeated for each of the four nucleotides, until the DNA sequence of the single stranded template is determined.[citation needed]
A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi, [Mathias Uhlen], and Pål Nyren.[non-primary source needed] In this alternative method, an additional enzyme, apyrase, is introduced to remove nucleotides that are not incorporated by the DNA polymerase.[citation needed] This enables the enzyme mixture— DNA polymerase, luciferase, and apyrase—to be added when sequencing is initiated, and kept in the reaction solution throughout the procedure (thus enabling easier automation).[citation needed] An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.[citation needed]
A third variant, a microfluidic pyrosequencing method, was described in 2005 by an industrial research team led by Jonathan Rothberg, at the company 454 Life Sciences.[non-primary source needed] This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support; Rothberg and co-workers demonstrated that sequencing could be performed in a highly parallel manner using a microfabrication and microarrays.[citation needed] This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.[citation needed] This first next generation sequencing instrument initiated a new era in genomics research,[according to whom?] and to rapidly falling prices for DNA sequencing,[according to whom?] allowing affordable whole genome sequencing.[citation needed]
"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.[better source needed][verification needed]
For the solution-based version of pyrosequencing, the single-strand DNA (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.[verification needed]
The process can be represented by the following equations: