Thermal denaturation of DNA molecules: A comparison of theory with experiment

Wartell, Roger M.; Benight, Albert S.

doi:10.1016/0370-1573(85)90060-2

Cited by 547 publications

(583 citation statements)

References 71 publications

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“…At low temperatures, all DNA exists in duplex form whereas the DNA is completely dissociated into single strands at high temperatures. The transition between these two states has been reported to span 2-48C (Wartell and Benight, 1985), with the midpoint denoted as melting temperature, T m . The sharpness of this transition depends on the length of the DNA molecule and other factors such as ionic strength.…”

Section: Denaturationmentioning

confidence: 99%

A kinetic model of quantitative real‐time polymerase chain reaction

Mehra

2005

Biotech & Bioengineering

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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.

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Section: Denaturationmentioning

confidence: 99%

A kinetic model of quantitative real‐time polymerase chain reaction

Mehra

2005

Biotech & Bioengineering

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“…The Poland-Scheraga model for DNA melting, in an Ising-type approach based on earlier ideas by Kittel [7] and Zimm [8], captures the competition between energetic stacking interactions of the intact double-stranded portion of the DNA molecule with the entropy gain from the more flexible ssDNA loops [3,[9][10][11][12][13]. 1 The statistical weight for the dissociation of base-pairs includes an enthalpic contribution H i j from unstacking base-pair i from base-pair j and an entropic contribution S i j for positioning of unbound bases.…”

Section: Poland-scheraga Free Energy One-bubble Casementioning

confidence: 99%

“…2 To interrupt the double helix to initiate a bubble, that is bordered by helical domains (compare Figure 2), co-operative interactions have to be overcome, to release base-stacked and paired nucleotides. This is expressed by an additional weight σ 0 ≡ exp(−γ 0 ), and varies between a few 10 −2 to 10 −5 , for different temperatures [11,12,17,18]. Finally, the formation of a flexible bubble includes an entropy loss corresponding to the returning probability of a random walk [3,[11][12].…”

Section: Poland-scheraga Free Energy One-bubble Casementioning

confidence: 99%

“…This is expressed by an additional weight σ 0 ≡ exp(−γ 0 ), and varies between a few 10 −2 to 10 −5 , for different temperatures [11,12,17,18]. Finally, the formation of a flexible bubble includes an entropy loss corresponding to the returning probability of a random walk [3,[11][12]. For DNA-bubbles, one usually assumes the form F(n) = (n + D) −c where the offset D accounts for persistence length effects [19], and c is the loop closure exponent [3,11,10].…”

Section: Poland-scheraga Free Energy One-bubble Casementioning

confidence: 99%

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Dynamic Approach to DNA Breathing

Metzler¹,

Ambjörnsson²

2005

J Biol Phys

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Abstract.Even under physiological conditions, the DNA double-helix spontaneously denatures locally, opening up fluctuating, flexible, single-stranded zones called DNA-bubbles. We present a dynamical description of this DNA-bubble breathing in terms of a Fokker-Planck equation for the bubble size, based on the Poland-Scheraga free energy for DNA denaturation. From this description, we can obtain basic quantities such as the lifetime, an important measure for the description of the interaction of a breathing DNA molecule and selectively single-stranded DNA binding proteins. Our approach is consistent with recent single molecule measurements of bubble fluctuation. We also introduce a master equation approach to model DNA breathing, and discuss its differences from the continuous Fokker-Planck description.

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“…The thermal stability of double-stranded nucleic acids reflects the local base composition of the nucleic acid and is influenced by several other parameters such as solvent conditions, especially ionic strength and concentration of derants, and concentration of the nucleic acid (for review of denaturation behaviour of nucleic acids and critical discussion of parameter values see [4][5][6][7]. The best method available for calculation of the thermal stability of a dsNA is based on an algorithm described by Poland (8) and on an accelerated version of that algorithm described by Fixman and Freire (9).…”

Section: Introductionmentioning

confidence: 99%