Calculation of Tm for Oligonucleotides

The melting temperature of an oligonucleotide (Tm) refers to the temperature at which the oligonucleotide is annealed to 50% of its exact complement. Accurate estimation of the Tm of an oligonucleotide is important for a wide variety of applications including PCR, hybridization, sequencing, and antigene targeting (antisense). In the absence of destabilizing agents such as urea or formamide, the Tm of an oligonucleotide will depend upon three major factors:

1. The oligonucleotide sequence: Generally, GC-rich sequences have a higher Tm than do AT-rich sequences. However, nearest-neighbor interactions also need to be taken into consideration to properly account for base stacking interactions.

2. The oligonucleotide concentration: High concentrations are more favorable for hybrid formation than are lower concentrations.

3. Salt concentration: The Tm increases with higher ionic strength due to the stabilizing effects of cations on DNA duplexes. This reflects the fact that cations bind preferentially to the duplex compared to the two separate single strands. Different cations may have different effects.

Historically, several methods have been provided for the estimation of Tm values. For short sequences that have 20 bases or less, the Wallace rule provides a first-order approximation¹:

_Tm = 2°C (A + T) + 4°C (G + C). (1)

Equation (1) assumes a salt concentration of 0.9 M, typical of dot blot and other hybridization assays.

Another equation based upon the GC content is the familiar expression from Howley et al.² in which T_m is given by

Tm = 81.5 + 0.41(%GC) – 500/L + 16.6 log[M], (2)

where L refers to the length of the oligonucleotide, and [M] is the concentration of monovalent cations. This formula is only applicable to long polynucleotide sequences. Neither Equation (1) nor (2) take into account nearest-neighbor interactions, i.e. base stacking effects.

The most accurate method of estimating the Tm of oligonucleotides, and that used at IDT, is based upon a thermodynamic analysis of the melting process from which it can be shown that

. (3)

The changes in enthalpy () and entropy () of duplex formation are calculated from nearest-neighbor thermodynamic parameters. R is the molar gas constant
(1.987 cal.K^-1mole^-1), and C is the molar concentration of oligonucleotide. A second term is added to Equation (3) to account empirically for the stabilizing effect of salt on the duplex:

. (4)

The linear dependence of the Tm on the logarithm of the salt concentration is illustrated by the experimental data in Figure 1. The effects of Na⁺ and K⁺ are equivalent within experimental error.

Figure 1. The dependence of Tm on salt concentration for a 30-mer (Lingyan Huang, unpublished results).

Traditionally, the nearest-neighbor parameter set of Breslauer has been used to estimate the Tm of oligonucleotide duplexes (3). Several alternative parameter sets have recently been reported^4-6. In agreement with the detailed analysis of Owczarzy et al.⁷, we have found that the Allawi and SantaLucia set of nearest-neighbor thermodynamic parameters⁵ provides the most accurate predictions of Tm. Therefore, we at IDT use this set of thermodynamic parameters to estimate Tm.

Studies in our laboratory have shown that the coefficient of 16.6 for the effect of salt on the Tm of long polynucleotides (Equation 2) is too high for oligonucleotides. A more accurate value in this case is 12.0. A similar value for oligonucleotides was suggested by SantaLucia, et al.⁴.

The Tm calculator on IDT's web site and the calculation of Tm given on our specification sheets are based on the nearest-neighbor thermodynamic parameter set of Allawi and SantaLucia⁵ and a coefficient of 12.0 for the salt dependence. We believe that this expression provides oligonucleotide users with the most accurate estimate of Tm currently available.

References

1. Wallace, R.B., Shaffer J., Murphy, R.F., Bonner, J., Hirose, T., and Itakura, K. (1979) "Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch."Nucleic Acids Research 6:3543-3547.



2. Howley, P.M., Israel, M.F., Law, M-F., and Martin, M.A. (1979) "A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes" J. Biological Chemistry 254:4876-4883.



3. Breslauer, K.J., Frank, R., Blocker, H., Marky, L.A. (1986) "Predicting DNA duplex stability from the base sequence." Proc.Natl. Acad. Sci. USA 83:3746-3750.



4. SantaLucia, Jr., J.S, Allawi, H.T., Seneviratne, P.A. (1996) "Improved nearest-neighbor parameters for predicting DNA duplex stability" Biochemistry 35:3555-3562.



5. Allawi, H.T., SantaLucia, J. Jr. (1997) "Thermodynamics and NMR of internal G.T mismatches in DNA." Biochemistry 36: 10581-10594.




6. Sugimoto, N., Nakano, S., Yoneyama, M., Honda, K. (1996) "Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes." Nucleic Acids Research. 24: 4501-4505.





7. Owczarzy, R., Vallone, P.M., Gallo, F.J., Paner, T.M., Lane, M.J., Benight,A.S. (1997) "Predicting sequence-dependent melting stability of short duplex DNA oligomers." Biopolymers 44:217-239