Melting temperatures of nucleic acids: discrepancies in analysis.

Melting temperature, T(m), is an important property of nucleic acid duplexes. It is typically determined from spectroscopic or calorimetric melting experiments. More than one analytical method has been used to extract T(m) values from experimental melting data. Unfortunately, different methods do not give the same results; the same melting data can be assigned different T(m) values depending upon which method is used to process that data. Inconsistencies or systematic errors between T(m)s reported in published data sets can be significant and add confusion to the field. Errors introduced from analysis can be greater than experimental errors, ranging from a fraction of degree to several degrees. Of the various methods, the most consistent and meaningful approach defines melting temperature as the temperature at the transition midpoint where half of the base pairs are melted and standard free energy is zero. Assuming a two-state melting behavior, we present here a set of general equations that can be used to reconcile these analytical T(m) differences and convert results to the correct melting temperatures at the transition midpoint. Melting temperatures collected from published sources, which were analyzed using different methods, can now be corrected for these discrepancies and compared on equal footing. The similar corrections apply to T(m) differences between calorimetric and spectroscopic melting curves. New algorithm for selection of linear sloping baselines, 2nd derivative method, is suggested, which can be used to automate melting curve analysis.