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The Principle of PyrosequencingTM Technology
TM
Pyrosequencing technology is a simple to use
technique for accurate and consistent analysis
of large numbers of short to medium length
DNA sequences.
Step 1
A sequencing primer is hybridized to a single stranded, PCR amplified, DNA
template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase,
luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS)
and luciferin.
Step 2
The first of four deoxyribonucleotide triphosphates (dNTP) is added to the
reaction. DNA polymerase catalyzes the incorporation of the deoxyribonucleotide triphosphate into the DNA strand, if it is complementary to the base
in the template strand. Each incorporation event is accompanied by release of
pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated
nucleotide.
Step 2
Polymerase
(DNA)n + dNTP
(DNA)n+1 + PPi
Step 3
Sulfurylase
light
APS+PPi ATP
luciferin oxyluciferin
Luciferase
Step 3
ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine
5´ phosphosulfate (APS). 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 seen as a
peak in a Pyrogram™. The height of each peak (light signal) is proportional to
the number of nucleotides incorporated.
ATP Light
time
nucleotide incorporation generates light
seen as a peak in the Pyrogram
Step 4
dNTP
Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and
unincorporated dNTPs. This switches off the light and regenerates the reaction
solution. The next dNTP is then added.
dNDP + dNMP + phosphate
Apyrase
ATP
Step 4
Apyrase
ADP + AMP + phosphate
Step 5
nucleotide sequence
G
C
G
C
–
A
GG
CC
T
T
A
G
C
T
Step 5
Addition of dNTPs is performed one at a time. It should be noted that
deoxyadenosine alfa-thio triphosphate (dATPaS) is used as a substitute for the
natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the
DNA polymerase, but not recognized by the luciferase.
As the process continues, the complementary DNA strand is built up and the
nucleotide sequence is determined from the signal peaks in the Pyrogram.
Technical Note 101
nucleotide added
Optimization of Pyrosequencing
technology
The Pyrosequencing reaction is simple and robust, using an
enzyme cascade system that gives unambiguous and
easily quantifiable results. Each component and parameter
has been optimized to achieve highly consistent results,
with accuracy levels of >99% for SNP genotyping, when
used routinely in automated systems from Pyrosequencing
AB.
• Substrate concentrations are optimized.
• Apyrase of a specific grade has been selected to ensure
that all dNTPs are degraded, including the alfa-thio-dATP
which is used instead of dATP. This enzyme also hydrolyzes ATP.
• The rate of dNTP degradation by apyrase is slower than
the rate of dNTP incorporation by the polymerase,
favouring sufficient incorporation of dNTPs.
• The rate of ATP synthesis by the sulfurylase is faster
than the rate of ATP hydrolysis by apyrase so that ATP
concentration and light production are in proportion to
the number of dNTPs incorporated.
• Reagents are carefully controlled to ensure adequate
activity and quality.
For further information and the latest applications
please visit www.pyrosequencing.com
Scientific references
Ronaghi, M. et al., (1998) A sequencing method based on real-time pyropohosphate,
a review. Science 281, 363-365
Nyrén et al., (1997) Detection of single-base changes using a bioluminometric primer
extension assay. Anal Biochem 244, 36-373
Ronaghi, M. et al., (1996) Real-time DNA sequencing using detection of pyrophosphate
release. Anal Biochem 242, 84-89
Nyrén, P., (1994) Apyrase immobilized on paramagnetic beads used to improve detection
limits in bioluminometric ATP monitoring. J Biolumin Chemilumin 9, 29-34
Nyrén P. et al., (1993) Solid phase DNA minisequencing by enzymatic luminometric
inorganic pyrophosphate detection assay. Anal Biochem 208, 171-175
Pyrosequencing, PSQ, Pyrogram and
The Solid Phase Sequencing method is covered by patents owned by Pyrosequencing AB and AB Sangtec Medical.
The PCR process is covered by several patents owned by Roche Molecular Systems and F. Hoffman-La Roche Ltd.
© Copyright 2002 Pyrosequencing AB.
are trademarks owned by Pyrosequencing AB. Pyrosequencing technology is covered by patents and patent applications owned by Pyrosequencing AB.
HEAD OFFICE:
Pyrosequencing AB
Vallongatan 1, SE-752 28 Uppsala, Sweden
Tel: +46 (0)18 56 59 00
Fax: +46 (0)18 59 19 22
[email protected], www.pyrosequencing.com
Pyrosequencing Inc. USA Massachusetts
Pyrosequencing Inc. USA California
Pyrosequencing Sarl. France
Pyrosequencing GmbH. Germany
Pyrosequencing AB. Nordic region
Pyrosequencing Ltd. UK & Eire
(877) 797-6767 (Toll Free)
(877) 797-6767 (Toll Free)
+33 (0)1 55 94 86 32
+49 (0)40 8195 7566
+46 (0)18 489 70 00
+44 (0)20 8334 8454
Printed in Sweden by Wikströms, Uppsala.
For research use only. Not intended for diagnostic purposes.
70-0001-6102 Aug 2002
Nyrén P., (1987) Enzymatic method for continuous monitoring of DNA polymerase
activity. Anal Biochem 167, 235-238