The Tm, or melting temperature, characterizes the stability of the DNA hybrid formed between an oligonucleotide and its complementary strand.

The Tm is critical for determining the optimal temperature to use an oligonucleotide as a primer in PCR application (annealing temperature), as a probe for in situ hybridization, and in Southern, Northern or Dot blot analyses.

In the absence of destabilizing agents, like formamide or urea, Tm will depend on 3 major parameters:

1. The sequence: a GC-rich sequence has a higher melting temperature.
2. The strand concentration: high oligonucleotide concentrations favor hybrid formation, which results in a higher melting temperature.
3. The salt concentration: high ionic strength results in a higher Tm as cations stabilize the DNA duplexes.

For oligonucleotides = 20 bases, we use the classical Tm = 2 x (A + T) + 4 x (G+C) calculation for melting temperature, according to Wallace rule for short oligonucleotides.

In the case of longer DNA fragments, the nearest-neighbor method (1-3), which combines solid thermodynamics and experimental data, offers a reliable estimation of the Tm.

This Tm calculation (4) is performed automatically by the GENSET SPARC workstation for every oligonucleotide produced, and it is printed on the label.

Here is the description of the Tm calculation by the nearest-neighbor method:

Tm =

H               (A + S) + Rln (Ct/4)

273.15 + 16.6 log [salt]

Where:

• H (cal mole-1) is the sum of the nearest-neighbor enthalpy changes for hybrid formation (<0).
  
  
• A (cal K-1mole-1) is a constant for helix initiation which is equal to -10.8 cal K-1mole-1 for nonself-complementary sequences and = - 12.4 for self-complementary sequences).
  
  
• S (cal K-1mole-1)is the sum of the nearest-neighbor entropy changes for hybrid formation (<0)
  
  
• R is the molar gas constant (1.987 cal K-1mole-1)
  
  
• Ct is the total molar concentration of strands when oligonucleotides are not self complementary or it is equal to 4 times this concentration in the case of self-complementary sequences.

In the nearest-neighbor formula, the two parameters affecting the Tm value are the salt concentration and the concentration of single-stranded DNA. These two experimental variables are choosen by Genset Oligos to optimize DNA amplification by PCR. These values are fixed at 50 mM salt and 250 pM oligonucleotide (5)

Reference

1. Breslauer K.J., Frank R., Blocker H., Markey L.A. (1986) Predicting DNA duplex stability from the base sequence - Proc. Natl. Acad. Sci. USA 83, 3746-3750
2. Freier S.M., Kierzek R., Jaeger J.A., Sugimoto N., Caruthers M.H., Nielson T., Turner D.H. (1986) Improved free-energy parameters for predictions of RNA duplex stabilit. - Proc. Natl. Acad. Sci. USA 83, 9373-9377
3. Schildkraut C., Lifson S. (1965) Dependence of the melting temperature of DNA on salt concentration - Biopolymers 3, 195-208
4. Rychlik W. - Oligo version 4.0, Reference Manual - National Biosciences, Inc., Plymouth, MN
5. Rychlik W., Spencer W.J., Rhoads R.E. - Nucleic Acids Res. 18, 6409-6412
6. Freier S.M. (1993) Hybridization: Considerations affecting Antisense Drugs in Antisense Research and Applications, eds. Grooke S.T. and Lebleu B. CRC Press, Inc., 67-82

The Tm value provided by Proligo is calculated for phosphodiester oligonucleotides used in DNA-amplification experiments. Even though this calculation does not take into account any nucleotide modification, like biotin, digoxigenin, fluorescent dyes, amine, it still gives you a good estimate of the starting conditions. In the case of phosphorothioates and RNA, reference (6) gives valuable information to calculate the Tm values.

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