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. 2004 Dec 2;32(21):e166.
doi: 10.1093/nar/gnh159.

A Multi-Enzyme Model for Pyrosequencing

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Free PMC article

A Multi-Enzyme Model for Pyrosequencing

Ali Agah et al. Nucleic Acids Res. .
Free PMC article

Abstract

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated.

Figures

Figure 1
Figure 1
The general principle of the Pyrosequencing reaction system.
Figure 2
Figure 2
Simulation versus experimental peak curves for (a) luciferase initiated by ATP dispensation, (b) luciferase/apyrase enzyme combination initiated by ATP dispensation, (c) luciferase/sulfurylase enzyme combination activity initiated by ATP dispensation, (d) luciferase/sulfurylase/apyrase enzyme combination initiated by ATP dispensation, (e) luciferase/sulfurylase/apyrase enzyme combination initiated by PPi dispensation. All cases are with 20 μg of luciferase. The variations in experimental peak heights are within ±5%.
Figure 3
Figure 3
Simulation and experimental results for peak height versus amount of luciferase in (ng) for (a) luciferase, (b) luciferase/apyrase, (c) luciferase/sulfurylase, (d) luciferase/sulfurylase/apyrase (ATP and PPi dispensation).
Figure 4
Figure 4
(a) Simulation versus experimental peak curve for polymerase/luciferase/sulfurylase/apyrase enzyme combination activity initiated by dNTP dispensation. (b) Peak height in arbitrary units versus DNA concentration in (pmol) for Pyrosequencing enzyme combination; simulation versus experimental data. The variations in experimental peak heights are within ±5%.
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Scheme 1. Pyrosequencing enzymatic reactions.
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Scheme 1. Pyrosequencing enzymatic reactions.
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Scheme 2. Simple reaction with forward and reverse constants.
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Scheme 2. Simple reaction with forward and reverse constants.
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Scheme 3. Simple enzymatic reaction.
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Scheme 3. Simple enzymatic reaction.
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Scheme 4. Detailed kinetic model of Klenow DNA polymerase.
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Scheme 4. Detailed kinetic model of Klenow DNA polymerase.
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Scheme 5. Proposed kinetic model for sulfurylase.
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Scheme 5. Proposed kinetic model for sulfurylase.
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Scheme 6. Detailed kinetic model of firefly luciferase.
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Scheme 6. Detailed kinetic model of firefly luciferase.
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Scheme 7. Proposed kinetic model for apyrase for (a) ATP (b) dNTP.

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