Pyrosequencing is a method of DNA sequencing based on the "sequencing by
synthesis" principle. It differs from Sanger sequencing, in that it relies on
the detection of pyrophosphate release on nucleotide incorporation, rather than
chain termination with dideoxynucleotides. The technique was
developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of
Technology in Stockholm in 1996. The desired DNA sequence is able to be
determined by light emitted upon incorporation of the next complementary
nucleotide by the fact that only one out of four of the possible AC/G nucleotides
are added and available at a time so that only one letter can be incorporated
on the single stranded template. The intensity of the light determines if
there are more than one of these "letters" in a row. The previous
nucleotide letter is degraded before the next nucleotide letter is added for
synthesis: allowing for the possible revealing of the next nucleotide(s) via
the resulting intensity of light. This process is repeated with each of the
four letters until the DNA sequence of the single stranded template is
determined. Procedure
"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
with another chemoluminescent enzyme. Essentially, the method allows
sequencing of 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.
The single-strand DNA 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 and
luciferin. The addition of one of the four
deoxynucleoside triphosphates initiates the second step. DNA polymerase
incorporates the correct, complementary dNTPs onto the template. This
incorporation releases pyrophosphate. ATP sulfurylase converts PPi to ATP in
the presence of adenosine 5´ phosphosulfate. This ATP acts as a
substrate for 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 camera and analyzed in a pyrogram.
Unincorporated nucleotides and ATP are degraded by the apyrase, and the
reaction can restart with another nucleotide.
Currently, a limitation of the method is that the lengths of individual reads of
DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the
800-1000 obtainable with chain termination methods. This can make the
process of genome assembly more difficult, particularly for sequences
containing a large amount of repetitive DNA. As of 2007, 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 and enzymatic template preparation. So Pyrosequencing can be
differentiated into two types, namely Solid Phase Pyrosequencing and Liquid
Phase Pyrosequencing. Commercialization
The company Pyrosequencing AB in Uppsala, Sweden was founded with venture
capital provided by HealthCap in order to commercialize machinery and reagents
for sequencing short stretches of DNA using the pyrosequencing technique.
Pyrosequencing AB was listed on the Stockholm Stock Exchange in 1999. It was
renamed to Biotage in 2003. The pyrosequencing business line was
acquired by Qiagen in 2008. Pyrosequencing technology was further
licensed to 454 Life Sciences. 454 developed an array-based pyrosequencing
technology which has emerged as a platform for large-scale DNA sequencing.
Most notable are the applications for genome sequencing and metagenomics. GS
FLX, the latest pyrosequencing platform by 454 Life Sciences, can generate 400
Mb in a 10-hour run with a single machine. Each run would cost about
5,000-7,000 USD. Hoffman LaRoche announced the
discontinuation of the 454 sequencing platform in 2013.
References = Further reading =
Metzker M.. "Emerging Technologies in DNA Sequencing". Genome Research 15:
1767–76. doi:10.1101/gr.3770505. PMID 16339375.