Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), 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 Mg(2+), deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO).

METHODS

The experimental T(m)s 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 [Mg(2+)], [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(square root of ([Mg2+]-[dNTPs])) (the concentrations in this formula are mmol/L) to correct DeltaS(0) and a DMSO term of 0.75 degrees C (%DMSO), the SE of the N-N T(m) estimate was 1.76 degrees C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula T(m) ( degrees C) = 77.1 degrees C + 11.7 x log[Na(+)(eq)] + 0.41(%GC) - 528/bp - 0.75 degrees C(%DMSO) gave a slightly higher SE of 1.87 degrees C. When all duplexes (matched and mismatched; n = 475) were included in N-N calculations, the SE was 2.06 degrees C.

CONCLUSIONS

This robust model, accounting for the effects of Mg(2+), DMSO, and dNTPs on oligonucleotide T(m) in PCR, gives reliable T(m) predictions using thermodynamic N-N calculations or empirical formulas.