A kinetic model of quantitative real‐time polymerase chain reaction

Abstract

Real-time polymerase chain reaction (PCR) is one of the most sensitive and accurate methods for quantifying transcript levels especially for those expressed at low abundance. The selective amplification of target DNA over multiple cycles allows its initial concentration to be determined. The amplification rate is a complex interplay of the operating conditions, initial reactant concentrations, and reaction rate constants. Experimentally, the compounded effect of all factors is quantified in terms of an effective efficiency, which is estimated by curve fitting to the amplification data. We present a comprehensive model of PCR to study the effect of various reactant concentrations on the amplification efficiency. The model is used to calculate the kinetic progression of the target DNA concentration with cycle number under conditions when different species are stoichiometrically or kinetically limiting. The reaction efficiency remains constant for the initial cycles. As the primer concentration becomes limiting, the efficiency is marked by a gradual decrease. This is in contrast to a steep decline under nucleotide limiting conditions. Under some conditions, commonly used experimentally, increasing primer concentration has the adverse effect of reducing the final amplified template concentration. This phenomenon seen at times experimentally is explained by the simulation results under rate limiting enzyme concentrations. Primer dimer formation is shown to significantly affect the reaction rates, effective efficiency, and the estimated initial concentrations. This model, by describing the interplay of the many operating variables, will be a useful tool in designing PCR conditions and evaluating its results. ß 2005 Wiley Periodicals, Inc.

Water Bridging Dynamics of Polymerase Chain Reaction in the Gauge Theory Paradigm of Quantum Fields

“…Long-range molecular correlations in the chemistry of DNA-enzyme interactions, discussed in Ref. [1], appear to play a key role in understanding how to reconcile the high efficiency and precise targeting of enzymatic catalytic activity with the intrinsically stochastic kinematics of a large number of molecular components [12][13][14]. The highly ordered pattern in the spatial molecular arrangements and the time ordered sequence of steps characterizing the catalytic activity indicate that biochemical methods and the traditional study of the dipole and multipole dynamical structure of the electronic quantum conformations, out of which the chemical properties of interacting molecules emerge, need to be supplemented by results in quantum field theory (QFT).…”

Adaption of SYBR Green-based reagent kit for real-time PCR quantitation of GC-rich DNA

“…The kinetic model of quantitative real-time PCR for normal DNA sequences has been well-documented (Gevertz et al, 2005;Mehra and Hu, 2005). Hence, additional studies are needed to elucidate the kinetic mechanism of amplification of GC-rich templates.…”

Dynamics and control of DNA sequence amplification

“…The most general control system is based on the sequenceand temperature-dependent state space model for DNA amplification described above. Other control systems are based on simplified PCR models previously proposed in the literature [22][23][24][25] and correspond to approximations to the model described in Section II. For these latter models, we only consider the previously developed model structures with rate constants estimated based on the above approach, since Marimuthu et al 14 have shown that the rate constants that were used in the previously proposed models are thermodynamically inconsistent.…”

“…The state equations of the melting, annealing, and extension reactions were solved independently. Also, the rate constants of the respective reactions were held constant 22 . We call this kind of PCR model a 'Staged' or 'On-Off' time invariant DNA amplification model, since the control vector fields are assumed to be either turned on or off at each step of a PCR cycle.…”