Pyrosequencing is a non-electrophoretic DNA sequencing (determining the order of nucleotides in DNA) method 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.
Principles<!-- I would transform this section in "History", imrpoving the chronology of discovery and commercialization of the method, while the detailed principle can go in the next session. -->
The principle of pyrosequencing was first described in 1993 by P. Nyrén, B. Pettersson, and M. Uhlen.<!--ONE CANNOT ESTABLISH A DISCOVERY FROM THE PRIMARY SOURCES OF A SUGGESTED DISCOVERER. SECONDARY SOURCES ARE ABSOLUTELY REQUIRED.--><!--Who was corresponding author? Also, no full names to appear until a source is presented that states them. Anal Bioch article only presents first initials.--> 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.<!--See note in all caps above.-->
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. 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. 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.
A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi, [Mathias Uhlen], and Pål Nyren.<!--See note in all caps above.-->In this alternative method, an additional enzyme, apyrase, is introduced to remove nucleotides that are not incorporated by the DNA polymerase. 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). An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.
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.<!--See note in all caps above.--> The pyrosequencing and other biomedical units of Biotage AB were sold to Qiagen in 2008. The 454 sequencing platform was replaced, in part, by Illumina dye sequencing, and by Applied Biosystems sequencing products.
Further reading
- For the corresponding PDF document, see this link.