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

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