The Principle of PyrosequencingTM Technology PyrosequencingTM technology is a simple to use technique for accurate and consistent analysis of large numbers of short to medium length DNA sequences. Step 1 Step 2 A sequencing primer is hybridized to a single stranded, PCR amplified, DNA Polymerase (DNA) + dNTP (DNA) + PPi template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, n n+1 luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin. Step 3 Step 2 The first of four deoxyribonucleotide triphosphates (dNTP) is added to the Sulfurylase reaction. DNA polymerase catalyzes the incorporation of the deoxyribo- light nucleotide triphosphate into the DNA strand, if it is complementary to the base APS+PPi ATP in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated luciferin oxyluciferin nucleotide. Luciferase Step 3 ATP Light ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine time 5´ phosphosulfate (APS). This ATP drives the luciferase mediated conversion of nucleotide incorporation generates light luciferin to oxyluciferin that generates visible light in amounts that are propor- seen as a peak in the Pyrogram tional 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 Step 4 peak in a Pyrogram™. The height of each peak (light signal) is proportional to dNTP Apyrase dNDP + dNMP + phosphate the number of nucleotides incorporated. Apyrase ATP Apyrase ADP + AMP + phosphate Step 4 Step 5 Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and nucleotide sequence unincorporated dNTPs. This switches off the light and regenerates the reaction GC – A GG CC T solution. The next dNTP is then added. 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. GGC TTA C As the process continues, the complementary DNA strand is built up and the nucleotide added nucleotide sequence is determined from the signal peaks in the Pyrogram. Technical Note 101 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 hydro- lyzes 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 Nyrén P., (1987) Enzymatic method for continuous monitoring of DNA polymerase activity. Anal Biochem 167, 235-238 For research use only. Not intended for diagnostic purposes. 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. Pyrosequencing, PSQ, Pyrogram and are trademarks owned by Pyrosequencing AB. Pyrosequencing technology is covered by patents and patent applications owned by Pyrosequencing AB. 70-0001-6102 Aug Aug 2002 70-0001-6102 Printed in Sweden by Wikströms, Uppsala. HEAD OFFICE: Pyrosequencing Inc. USA Massachusetts (877) 797-6767 (Toll Free) Pyrosequencing AB Pyrosequencing Inc. USA California (877) 797-6767 (Toll Free) Vallongatan 1, SE-752 28 Uppsala, Sweden Pyrosequencing Sarl. France +33 (0)1 55 94 86 32 Tel: +46 (0)18 56 59 00 Pyrosequencing GmbH. Germany +49 (0)40 8195 7566 Fax: +46 (0)18 59 19 22 Pyrosequencing AB. Nordic region +46 (0)18 489 70 00 [email protected], www.pyrosequencing.com Pyrosequencing Ltd. UK & Eire +44 (0)20 8334 8454.