Best practice for improved accuracy: A critical reassessment of van't Hoff analysis of melt curves

Published

June 7, 2022

Author(s)

Jacob Majikes, Michael Zwolak, James Alexander Liddle

Abstract

Biomolecular, particularly DNA, thermodynamics are frequently determined via van't Hoff analysis of optically-measured melt curves. The popularity of this approach is a result of both the ready availability of high resolution melt (HRM) curve data as a byproduct of other experiments, such as those using polymerase chain reaction (PCR), or because the molecular mass of the system is so great, e.g., DNA origami, that it precludes the use of calorimetric methods. Accurate and precise values of thermodynamic parameters are essential for the modeling of complex systems, such as RNA tertiary structure and DNA origami folding because the errors associated with each motif when constructing a folding energy landscape can compound. This can significantly reduce the power of predictive models. We follow the sources of uncertainty as they propagate through a typical van't Hoff analysis to build best practices for melt/anneal experiments and subsequent data analysis. We do, however, assume a perfect reporter baseline correction. We find that van't Hoff analysis is highly sensitive to certain experimental protocols. With appropriately designed experiments and analysis, van't Hoff fitting can provide surprisingly high precision, using even basic equipment, e.g. enthalpy precision as low as a hundredth of a kJ.mol-1 for an 8 base DNA oligomer.

Citation

Biophysical Journal

Volume

121

Issue

11

Pub Type

Journals

Download Paper

https://doi.org/10.1016/j.bpj.2022.05.008

Local Download

Keywords

DNA, Thermodynamics

Citation

Majikes, J. , Zwolak, M. and Liddle, J. (2022), Best practice for improved accuracy: A critical reassessment of van't Hoff analysis of melt curves, Biophysical Journal, [online], https://doi.org/10.1016/j.bpj.2022.05.008 , https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=932261 (Accessed October 6, 2024)