title: Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas.
abstract: 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.
authors.affiliation: Department of Clinical Chemistry, Georg-August University, Robert Koch Strasse 40, 37075 Goettingen, Germany. nahsen@gwdg.de
authors.forename: N
authors.initials: N
authors.lastname: von Ahsen
authors.forename: C T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: E
authors.initials: E
authors.lastname: Schütz
mesh_terms: D002413:Cations, Divalent; D003854:Deoxyribonucleotides; D004121:Dimethyl Sulfoxide; D007202:Indicators and Reagents; D008274:Magnesium; D008433:Mathematics; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D002413:Cations, Divalent; D003854:Deoxyribonucleotides; D007202:Indicators and Reagents; D009841:Oligonucleotides; D008274:Magnesium; D004121:Dimethyl Sulfoxide