Melting Temperature (Tm) Calculation for Oligonucleotides
and Oligonucleotides containing Bridged Nucleic Acids (BNAs)
Several mutation detection techniques rely on the chemical- or temperature-driven melting behavior of short oligonucleotides that are used for the hybridization of PCR-amplified DNA. These techniques include temperature gradient gel electrophoresis (TGGE) or denaturing gradient gel electrophoresis (DGGE), DNA blotting and hybridization probe assays and many others. The design of synthetic probes that increase the stability of the dsDNA oligonucleotids makes the use of these techniques possible. The influence of mismatches on dsDNA stability is based on thermodynamic principles which underlying physico-chemical mechanisms have been investigated extensively in the last decade. The stability of a single base pair binding is influenced by the surrounding nearest neighbor base pairs. The thermodynamic parameters for every matched or mismatched base pair were experimentally defined and published and allow for the prediction of oligonucleotide duplex stability with or without mismatches.
What is the melting or annealing temperature of a DNA or RNA molecule?
The melting or annealing temperature (Tm) of a DNA or RNA molecule is the temperature at which a DNA, RNA or DNA/RNA double helix dissociates into single strands and where half the molecules are double stranded and half are single stranded.
The Tm reflects the stability of DNA or RNA duplexes. More stable complexes will have higher melting temperatures. Experimental conditions such as salt and oligonucleotide concentrations will affect the hybridization process therefore the Tm needs to be measured under standard conditions.
In general, longer strands have higher melting temperatures, as do sequences with higher G and C content. The best estimate of the oligonucleotide melting temperature is the use of thermodynamic parameters for the calculations. These estimates are useful for evidence based design of molecular biological experiments in which the melting behavior is important. Values for entropy and enthalpy of Watson-Crick pairs have been determined by many researchers in the past and can be found in the literature. SantaLucia in 1998 published enthalpy and entropy based unified values for the reliable prediction of Tm reflecting the DNA duplex stability. Since the Tm of the duplex is also influenced by the concentration of the oligonuleotides used in an experiment experimentally derived correction equations are also needed. For longer sequences alternative methods to estimate melting temperatures for duplex stability were developed as well. These methods are based on the length and GC content of the oligonucleotides.
Reliable calculations should allow for the design of oligonucleotide probes allowing the discrimination of wild-type and mutant sequences by designing probes with the proper Tm.
Good sequencing primers or hybridization probes should have the following characteristics:
- They need to form stable duplexes with the target sequence under the experimental conditions used;
- Be highly specific for the intended target sequence, and no form base-pairs to other regions within the template; and
- Very importantly, the sequences should not anneal to themselves.
The formation of stable duplexes is especially important if the oligonucleotide probe is used for screening of complex DNA libraries. High specificity and not annealing to itself are important c parameters needed for both screening and sequencing.
Computerized methods have made the search for an optimal oligonucleotide which would meet all three of these criteria less laborious. Furthermore, calculating the duplex dissociation temperature is critical to characterize oligonucleotides.
The dissociation temperature (Td) can be calculated with the following formula:
Td = 2°C x number of AT bp + 4°C x number of GC bp
The “nearest neighbor model” allows for the use of nearest neighbor thermodynamic values with the following equation:
Td = [ΔH / ΔS + R x ln(Ct/4)] – 273.15 °C –t
Where ΔH and ΔS are the enthalpy and entropy for helix formation. R is the molar gas constant [1.987 (cal/°C x mol)], and Ct is the concentration of the probe. The constant t is a temperature correction for filter hybridization with a value equal to 7.6 °C.
The melting temperature (Tm) of a duplex can be calculated with the following equation:
This model also takes into account the influence of salts present in the sample. The next equation shows how the Tm can be estimated in the presence of sodium ions.
Reference: Rychlik and Rhoads in Nucleic Acid Research 1989, 17, 21, 8543-8551.
Determination of the melting point
The common way to determine the actual melting point of an oligonucleotide duplex is to use a cell or quartz cuvette in a thermostat as part of a UV spectrometer. The temperature is plotted versus the absorbance resulting in an S-shaped curve with two observed plateaus. The absorbance reading halfway between the plateaus correspond to the Tm.
A real-time PCR instrument allows the determination of the Tm as well, for example, using SYBR green to stain the resulting PCR products.
Testing different web based oligonucleotide calculators for consistency in results
Chavali et al. in 2005 published a paper in the Journal Bioinformatics in which the researchers reported their findings for their study in which they compared different oligonucleotide calculators for their ability to predict the best melting temperature with the least deviation.
The study was divided into three sections.
- The identification of the best oligonucleotide properties calculator to predict the best Tm to allow for the calculation of the optimal annealing temperature for PCR amplifications.
- The evaluation of the secondary structure predictions.
- Testing the efficiency of primer designing software and identifying the best one.
Experimental methods employed were thermal melting studies, Tm predictions, statistical analysis, and calculation of optimal annealing temperature, secondary structure studies and primer design studies. In this study the Tm of 108 oligonucleotides was predicted using 25 oligonucleotide properties calculators. Tm deviation values in the range of 7°C were observed.
Conclusion
These results indicate that the theoretical methods are good to estimate the Tm but may not be very accurate. Furthermore, it is advisable to verify the predicted results experimentally for selected oligos when high throughput methods are used.
Note 1: A precise optimum annealing temperature must be determined empirically.
Most of the calculator software use similar algorithms and models for more details please review the paper.
Note 2: Prediction of Tm value of BNA/DNA chimeric oligonucleotides.
The R&D group at BSI has recently developed our own Tm value prediction calculator based on a modified nearest-neighbor themodynamic model and experimental data. To obtain accurate Tm value prediction, oligonucleotides ranging from 8 to 20 nucleotides are recommended. Currently for our customers, we are providing the predicted Tm values. If the predicted Tm value is needed for your research, please contact info@biosyn.com.
Reference:
Chavali et al. in Bioinformatics. 2005 Oct 15;21(20): 3918-25. Epub 2005 Aug 16.
Glossary
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Tm
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Melting temperature: The melting temperature is the temperature in °C at which 50% of the oligonucleotide and its perfect complement are in a duplex.
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Td
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Dissociation temperature: The dissociation temperature is the temperature in °C at which 50% of an oligonucleotide and its perfect filter-bound complement are in duplex at the particular salt concentration and total strand concentration.
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ΔH
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Enthalpy: The Enthalpy change can be calculated by subtracting the bond energies of the product from the bond energies of the reactions.
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ΔS
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Entropy: The change in entropy is the tendency for randomness in a system. Natural systems have the tendency toward low enthalpy and high entropy.
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