Effects of Sodium Ions on DNA Duplex Oligomers:  Improved Predictions of Melting Temperatures

Abstract

Melting temperatures, T(m), were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between T(m) and ln [Na(+)] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 degrees C when tested against an independent validation set of T(m) measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this T(m) salt correction is insensitive to DNA concentration. The T(m) salt correction function was found to be sequence-dependent and varied with the fraction of G.C base pairs, in agreement with previous studies of genomic and polymeric DNAs. The salt correction function is independent of oligomer length, suggesting that end-fraying and other end effects have little influence on the amount of sodium counterions released during duplex melting. The results are discussed in the context of counterion condensation theory.

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

“…DNA concentrations were determined from absorbance (33). Complementary single-strands were mixed in a 1:1 molar ratio.…”

“…Spectrophotometer software was modified to improve resolution and to more finely control rate of temperature changes (33). Both denaturation and renaturation transition curves were collected, and temperature was increased linearly at a rate of 24.9 ( 0.3°C/h.…”

“…Melting curves of buffers alone were subtracted from raw DNA melting curves. Regions of linear increase of absorbance before and after the melting transition were least-squares fitted to lower and upper linear baselines, and the fraction of broken base pairs, θ, was calculated as described previously (33). All melting curves showed a single, S-shaped transition for all buffers studied, allowing T m to be unambiguously determined (see Figure S1 of the Supporting Information).…”

“…Twelve DNA duplexes were melted in buffers containing 10 mM Tris and KCl of various concentrations ranging from 50 mM to 1 M as well as in sodium buffers (33). The stabilizing effect of K + ions is also sequence dependent, and the dependence of 1/T m on ln [K + ] is nonlinear.…”

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

“…The use of different tar-gets is important because even though the FISH methodology is more frequently described for RNA targets, there are also several studies using modified probes which use DNA as a target, e.g., (Celeda et al 1994;Matthiesen and Hansen 2012). In accordance with the literature, a higher Tm was observed at higher salt-concentrated solutions (Owczarzy et al 2004). Higher concentrations of NaCl allow the repulsion between the negatively charged oligonucleotides to be increasingly compensated by the positive counterions.…”