Sequence-Dependent Biophysical Modeling of DNA Amplification

Abstract

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.

Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

“…In such a case interplay of this equilibrium with the processes of polymerase association and dissociation with the template-primer complex and with the polymerization of the DNA chain should be taken into account. However, as shown by Marimuthu et al (2014), the rate contant of primer dissociation grows rapidly with the temperature, while the association rate constant varies very weakly. Therefore, the equilibrium constant of primer dissociation, i.e., the instability of the primer-template complex, grows strongly with temperature.…”

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

“…The identification and quantification of PCR enhancers are important to understand activities of commercial buffers and eventually improve their performance. The effects of cosolvents on the PCR amplification can be studied according to quantitative models for PCR amplification [1,19]. These models show that the optimal co-solvent concentration corresponds to that where the favorable effects of the co-solvent on DNA melting and secondary structure outweigh the unfavorable effects on polymerase stability and activity with the greatest margin; also, the maximal cosolvent concentration compatible with PCR generally corresponds to that beyond which the polymerase activity is extinguished.…”

Absolute quantification of real-time PCR data with stage signal difference analysis

“…Discrete cycle numbers using recursive deterministic reaction model were also developed to simulate and fit the qPCR data globally [21]. Sequence variations were analyzed with a thermodynamic deterministic model [22]. These results yield insights into the mechanism of qPCR reactions, but none of them give the clues about why log-linear relationship emerges.…”