RT Journal Article
SR Electronic
T1 Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg^{2+}, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas
JF Clinical Chemistry
JO Clin. Chem.
FD American Association for Clinical Chemistry
SP 1956
OP 1961
VO 47
IS 11
A1 von Ahsen, Nicolas
A1 Wittwer, Carl T.
A1 Schütz, Ekkehard
YR 2001
UL http://clinchem.aaccjnls.org/content/47/11/1956.abstract
AB Background: Many techniques in molecular biology depend on the oligonucleotide melting temperature (Tm), and several formulas have been developed to estimate Tm. Nearest-neighbor (N-N) models provide the highest accuracy for Tm 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 Tms 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(\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\sqrt{{[}Mg^{2{+}}{]}\ {-}\ {[}dNTPs{]}}\) \end{document}) (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 Tm estimate was 1.76 °C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula Tm (°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 Tm in PCR, gives reliable Tm predictions using thermodynamic N-N calculations or empirical formulas.