Melting Temperature

Melting temperature (TM) is the temperature at which an oligonucleotide duplex is 50% in single-stranded form and 50% in double-stranded form. IDT's online Oligo Analyzer estimates TM from the nearest-neighbor two-state model, which is applicable to short DNA duplexes,

Tm Equation OligoAnalyzer

where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameters calculated from the sequence and the published nearest neighbor thermodynamic parameters, R is the ideal gas constant (1.987 cal· K-1mole-1), [oligo] is the molar concentration of an oligonucleotide, and the constant of -273.15 converts temperature from Kelvin to degrees of Celsius. The most accurate, nearest-neighbor parameters were obtained from the following publications for DNA/DNA base pairs (Allawi, H., SantaLucia, J.,Jr., Biochemistry, 36, 10581), RNA/DNA base pairs (Sugimoto, N. et al., Biochemistry, 34, 11211), RNA/RNA base pairs (Xia, T. et al., Biochemistry, 37, 14719), and LNA/DNA base pairs (McTigue, P.M. et al., Biochemistry, 43, 5388).

TM calculations for oligonucleotides containing non-consecutive, isolated LNA nucleotides hybridized to a DNA template utilize LNA energetic parameters from McTigue, P.M. et al. All other LNA nucleotides (i.e., consecutive LNA bases on a DNA template or any LNA nucleotides on an RNA template) are approximated because nearest-neighbor parameters for these types of bases pairs have yet to be published. Applications requiring extremely accurate predictions of the TM for LNA containing oligonucleotides should be reviewed with a technical support representative from Exiqon, Inc. (


Table 1: Parameters for Equation Above in Reciprocal Kelvins

Parameter Value (K-1) Standard Error (K-1)
a 3.92 x 10-5 0.2 x 10-5
b -9.11 x 10-6 0.5 x 10-6
c 6.26 x 10-5 0.4 x 10-5
d 1.42 x 10-5 0.08 x 10-5
e -4.82 x 10-4 0.7 x 10-4
f 5.25 x 10-4 0.2 x 10-4
g 8.31 x 10-5 0.2 x 10-5