Numerical Simulations of EXPAR Reaction Kinetics Using COPASI Software
Authors:
Chelsea Edirisuriya, Stephanie KimMentor:
Angelika Niemz, Associate Professor, Keck Graduate InstituteNucleic acid amplification is important in many research and clinical diagnostics applications. Nucleic acid amplification through the EXPonential Amplification Reaction (EXPAR) can be performed at a single reaction temperature using simple instrumentation, unlike the Polymerase Chain Reaction (PCR), which involves thermocycling and complex equipment. The project goal was to better understand the coupled biochemical reactions involved in EXPAR, thereby facilitating assay optimization. We programmed the conventional and an alternative model for EXPAR into a computer software called COPASI, to numerically model the reaction time course and transient concentrations of intermediate species. In the conventional EXPAR reaction model, a trigger DNA strand binds to a single-stranded DNA template, is extended by the polymerase, a nicking enzyme cuts the top strand, which releases a new copy of the trigger sequence, followed by the next round of extension, cut, and release. Newly formed triggers activate additional template sequences, leading to exponential amplification. The COPASI model for the conventional model generally agrees with experimental data, and enabled us to understand how changes in the nicking enzyme and polymerase concentrations, and kcat values dictate the rate limiting step in EXPAR. However, we hypothesize that the reaction in reality follows an alternative model, wherein the polymerase binds first to the single-stranded DNA template, followed by trigger binding, extension, cut, and release, as above. This alternative model can better explain non-specific amplification observed in EXPAR. We hypothesize that during non-specific amplification, polymerase bound to the single-stranded template can prime DNA extension from a single dNTP present in the active site, rather than an entire trigger sequence. We have programmed the alternative model for specific amplification with the trigger into COPASI, although analysis of COPASI results is still in progress. In the future, we will expand the model to also include non-specific amplification in the absence of trigger.