UV-Melting Curves: Difference between revisions
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One traditional method to determine the stability of DNA or RNA duplexes is to heat an aqueous solution of the duplex in question and observing the change in the UV absorption of the solution as the temperature increases. Upon dissociation of the duplex, the base stacking is lost, so that the UV absorption increases. Plotting the UV absorption against the temperature then yields a so-called melting curve. The temperature at which half of the duplex is dissociated is called the 'melting point'. The more stable the duplex, the higher the UV-melting point. Standard settings are a heating rate of 1 °C/min and UV monitoring at 260 nm. From the UV melting curves, enthalpy and entropy of duplex formation/dissociation can be derived. | One traditional method to determine the stability of DNA or RNA duplexes is to heat an aqueous solution of the duplex in question and observing the change in the UV absorption of the solution as the temperature increases. Upon dissociation of the duplex, the base stacking is lost, so that the UV absorption increases. Plotting the UV absorption against the temperature then yields a so-called melting curve. The temperature at which half of the duplex is dissociated is called the 'melting point'. The more stable the duplex, the higher the UV-melting point. Standard settings are a heating rate of 1 °C/min and UV monitoring at 260 nm. From the UV melting curves, enthalpy and entropy of duplex formation/dissociation can be derived. | ||
Typically, the point of inflection of the first derivative of the (often smoothed) experimental curve is being interpreted as the UV melting point. To avoid misinterpretation of experimental data, it is recommended to measure melting curves at extinction values of between 01. and 1.2. Further, only sigmoidal transitions should be interpreted, and hyperchromicities accompanying the melting transition should be in the range of 8-35% of the initial extinction reading. Also, it is important to establish proper baselines in the low and high temperature region of the curve. Finally, evaporation effects should be avoided, e.g. by ensuring a properly filled and sealed cuvette for the | Typically, the point of inflection of the first derivative of the (often smoothed) experimental curve is being interpreted as the UV melting point. To avoid misinterpretation of experimental data, it is recommended to measure melting curves at extinction values of between 01. and 1.2. Further, only sigmoidal transitions should be interpreted, and hyperchromicities accompanying the melting transition should be in the range of 8-35% of the initial extinction reading. Also, it is important to establish proper baselines in the low and high temperature region of the curve. Finally, evaporation effects should be avoided, e.g. by ensuring a properly filled and sealed cuvette for the acquisition of UV-melting curves. | ||
Revision as of 07:17, 2 July 2024
UV-Melting Points
One traditional method to determine the stability of DNA or RNA duplexes is to heat an aqueous solution of the duplex in question and observing the change in the UV absorption of the solution as the temperature increases. Upon dissociation of the duplex, the base stacking is lost, so that the UV absorption increases. Plotting the UV absorption against the temperature then yields a so-called melting curve. The temperature at which half of the duplex is dissociated is called the 'melting point'. The more stable the duplex, the higher the UV-melting point. Standard settings are a heating rate of 1 °C/min and UV monitoring at 260 nm. From the UV melting curves, enthalpy and entropy of duplex formation/dissociation can be derived.
Typically, the point of inflection of the first derivative of the (often smoothed) experimental curve is being interpreted as the UV melting point. To avoid misinterpretation of experimental data, it is recommended to measure melting curves at extinction values of between 01. and 1.2. Further, only sigmoidal transitions should be interpreted, and hyperchromicities accompanying the melting transition should be in the range of 8-35% of the initial extinction reading. Also, it is important to establish proper baselines in the low and high temperature region of the curve. Finally, evaporation effects should be avoided, e.g. by ensuring a properly filled and sealed cuvette for the acquisition of UV-melting curves.
References
Overview
Breslauer, K.J. Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order-disorder transitions. Methods Enzymol. 1995, 259, 221-242.
Paper mentioning the Meltwin software for extracting enthalpy and entropy from melting curves
McDowell, J.A.; Turner, D.H. Investigation of the structural basis for thermodynamic stabilities of tandem GU mismatches: solution structure of (rGAGGUCUC)2 by two-dimensional NMR and simulated annealing. Biochemistry 1996, 35, 14077-14089.
Application of melting curves for DNA duplexes
C. Ahlborn, K. Siegmund, C. Richert, Isostable DNA. J. Am. Chem. Soc. 2007, 129, 15218-15232.