Published online 2 December 2004
Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved
A multi-enzyme model for pyrosequencing
1 Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA, 2 Center for Integrated Systems, Stanford University, Palo Alto, CA, USA and 3 Institute for Biochemistry and Biophysics, Tehran University, Iran
* To whom correspondence should be addressed. Tel: +1 650 812 1971; Fax: +1 650 812 1975; Email: mostafa{at}stanford.edu
Received May 25, 2004; Revised August 25, 2004; Accepted October 30, 2004
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 KM and kcat of all four enzymes in Pyrosequencing can be calculated.