A C G T

Cascade Reaction
Chemistry
P Y R O S E Q U E N C I N G – O N E H O U R TO E X P L I C I T S E Q U E N C E D ATA

The principle of PyrosequencingTM

• True sequence information
• Fully quantitative genotyping
• Easy to read and share
• Suitable for clinical research

light

time

Pyrosequencing™ is an established genetic analysis method based on the

principle of sequencing by synthesis. It is the only genetic analysis method capable of
delivering explicit sequence information within minutes.
Pyrosequencing is an ideal choice for genetic analysis in clinical research. The output data
from Pyrosequencing is the gold standard of genetic information: real sequence data. This
is the best possible assurance of a correct genetic test.

Pyrosequencing is brought to you by Biotage, a global company focused on life

sciences. More information at www.biotage.com
Pyrosequencing chemistry, step by step

Step 1
A sequencing primer is hybridized to a single stranded, PCR amplified, DNA
template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, Step 2
luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate Polymerase
(DNA)n + dNTP (DNA)n+1 + PPi
(APS) and luciferin.

Step 2
The first of four deoxyribonucleotide triphosphates (dNTP) is added to the Step 3
reaction. DNA polymerase catalyzes the incorporation of the deoxyribo-
nucleotide triphosphate into the DNA strand, if it is complementary to the Sulfurylase
base in the template strand. Each incorporation event is accompanied by light
release of pyrophosphate (PPi) in a quantity equimolar to the amount of APS+PPi ATP
incorporated nucleotide.
luciferin oxyluciferin

Step 3 Luciferase

ATP sulfurylase quantitatively converts PPi to ATP in the presence of ad-

ATP Light
enosine 5´ phosphosulfate (APS). This ATP drives the luciferase mediated
time
conversion of luciferin to oxyluciferin that generates visible light in amounts
that are proportional to the amount of ATP. The light produced in the lucifer- nucleotide incorporation generates light
seen as a peak in the Pyrogram
ase-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. Step 4
Apyrase
dNTP dNDP + dNMP + phosphate

Step 4 ATP
Apyrase
ADP + AMP + phosphate
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. Step 5
nucleotide sequence
G C – A GG CC 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

G C T A G C T
and the nucleotide sequence is determined from the signal peaks in the
Pyrogram. nucleotide added

70-0001-6104 December 2004

PyrosequencingTM systems are designed for Laboratory Use Only

which means that they may be used for either research purposes or by high complexity CLIA certified labs.

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