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 oligonucleot ide melting temperature (T-m), and several formulas have been developed to estimate Tm. 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 eff ects of reagents commonly used in PCR, such as Mg2+, deoxynucleotide tripho sphates (dNTPs), and dimethyl sulfoxide (DMSO). Methods: The experimental T(m)s of 475 matched or mismatched target/probe d uplexes were obtained in our laboratories or were compiled from the literat ure based on studies using the same real-time PCR platform. This data set w as used to evaluate the contributions of [Mg2+], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical form ulas based on GC content, length, and the equivalent sodium ion concentrati on of cations [Na-eq(+)] were obtained by multiple regression. Results: When we used [Na-eq(+)] = [Monovalent cations] + 120(root [Mg2+] - [dNTPs]) (the concentrations in this formula are mmol/L) to correct DeltaS (0) and a DMSO term of 0.75 degreesC (%DMSO), the SE of the N-N T-m estimat e was 1.76 degreesC for perfectly matched duplexes (n = 217). Alternatively , the empirical formula T-m (degreesC) = 77.1 degreesC + 11.7 X log[Na-eq()] + 0.41(%GC) - 528/bp - 0.75 degreesC(%DMSO) gave a slightly higher SE of 1.87 degreesC. When all duplexes (matched and mismatched; n = 475) were in cluded in N-N calculations, the SE was 2.06 degreesC. Conclusions: This robust model, accounting for the effects of Mg2+, DMSO, a nd dNTPs on oligonucleotide Tm in PCR, gives reliable T. predictions using thermodynamic N-N calculations or empirical formulas. (C) 2001 American Ass ociation for Clinical Chemistry.