Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas

Background: Many techniques in molecular biology depend on the oligonucleotide melting temperature (T_m), and several formulas have been developed to estimate T_m. Nearest-neighbor (N-N) models provide the highest accuracy for T_m prediction, but it is not clear how to adjust these models for the effects of reagents commonly used in PCR, such as Mg²⁺, deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO). Methods: The experimental T_ms of 475 matched or mismatched target/probe duplexes were obtained in our laboratories or were compiled from the literature based on studies using the same real-time PCR platform. This data set was used to evaluate the contributions of [Mg²⁺], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical formulas based on GC content, length, and the equivalent sodium ion concentration of cations [Na⁺_eq] were obtained by multiple regression. Results: When we used [Na⁺_eq] = [Monovalent cations] + 120(⁠ \(\sqrt{{[}Mg^{2{+}}{]}\ {-}\ {[}dNTPs{]}}\) ⁠) (the concentrations in this formula are mmol/L) to correct ΔS⁰ and a DMSO term of 0.75 °C (%DMSO), the SE of the N-N T_m estimate was 1.76 °C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula T_m (°C) = 77.1 °C + 11.7 × log[Na⁺_eq] + 0.41(%GC) − 528/bp − 0.75 °C(%DMSO) gave a slightly higher SE of 1.87 °C. When all duplexes (matched and mismatched; n = 475) were included in N-N calculations, the SE was 2.06 °C. Conclusions: This robust model, accounting for the effects of Mg²⁺, DMSO, and dNTPs on oligonucleotide T_m in PCR, gives reliable T_m predictions using thermodynamic N-N calculations or empirical formulas.