Dynamics and control of DNA sequence amplification

Marimuthu, Karthikeyan; Chakrabarti, Raj

doi:10.1063/1.4899053

Cited by 4 publications

(4 citation statements)

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“…In reference, a nonlinear PCR amplification model was proposed, which relates polymerase activity and thermostability as well as DNA melting temperature to PCR product yield and Cq value. The proposed model can be used to predict the Cq value as a function of cosolvent concentration, given the effects of cosolvent on each of these three properties and thus to identify the optimal cosolvent concentration for the amplification of a given template with a characterized polymerase enzyme . Furthermore, it was proposed that activity decline and thermal denaturation, by cosolvent, increase the minimum extension time, which in turn decrease product yield. , In particular, the cosolvent concentration at which activity is extinguished determines the effective range of cosolvent concentrations because of the greater effect of the cosolvent on the enzyme activity at those concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…We note that analogous methods can, in principle, be applied to DNA polymerases from diverse families, including any other template-dependent DNA polymerase and with minor modifications, also family X polymerases such as the terminal deoxynucleotidyl transferase (TdT) polymerase that are being engineered for enzymatic DNA synthesis applicationsand for which cosolvent stability has been recently identified as an important design objective . Finally, optimal PCR cycling protocols for these engineered polymerases in aqueous-organic mediaincluding the use of lower denaturation temperatures, lower extension temperatures, and optimal times for each PCR stepcan be determined using optimization techniques previously introduced. , …”

Section: Discussionmentioning

confidence: 99%

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Evolution of Organic Solvent-Resistant DNA Polymerases

Elias,

Guan,

Hudson

et al. 2023

ACS Synth. Biol.

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The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evolution of Organic Solvent-Resistant DNA Polymerases

Elias,

Guan,

Hudson

et al. 2023

ACS Synth. Biol.

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“…To overcome such problem, a coevolution of the existing species and their fitness landscape is desirable [15][16][17][18]. Another subject of major interest in evolutionary biology is the fact that evolution is similar to an optimization process: a population evolves in time in order to adapt as much as possible to some external conditions [19][20][21]. A final relevant aspect in evolution is understanding the role played by information, that is, to formulate rigorously the process of adaptation as a learning mechanism.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamics and evolutionary biology through optimal control

Bravetti

Padilla

2019

Automatica

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We consider a particular instance of the lift of controlled systems recently proposed in the theory of irreversible thermodynamics and show that it leads to a variational principle for an optimal control in the sense of Pontryagin. Then we focus on two important applications: in thermodynamics and in evolutionary biology. In the thermodynamic context, we show that this principle provides a dynamical implementation of the Second Law, which stabilizes the equilibrium manifold of a system. In the evolutionary context, we discuss several interesting features: it provides a robust scheme for the coevolution of the population and its fitness landscape; it has a clear informational interpretation; it recovers Price equation naturally; and finally, it extends standard evolutionary dynamics to include phenomena such as the emergence of cooperation and the coexistence of qualitatively different phases of evolution, which we speculate can be associated with Darwinism and punctuated equilibria. * alessandro.bravetti@cimat.mx †

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“…We also present simulations and new experimental results in support of this framework, including identification of the mechanism of a compound that activates the human SIRT3 enzyme, which does not contain an allosteric site, under physiologically relevant conditions. Methods from metabolic control theory 4 , which include the control of biochemical reaction network dynamics 5 , can be used to determine when overexpression or activation of an enzyme will increase flux through the associated reactive pathway. Typically, overexpression or activation of an enzyme will not increase reactive pathway flux.…”

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confidence: 99%