About the Calculations
None of the following melting temperature (Tm) calculations provide an adjustment for magnesium or manganese divalent cation concentration, although lack of these cations has been shown to adversely affect proper duplex formation. See Nakano et al, (1999) Proc. Nuclec Acids Res. 27:2957-65. (Abstract)
Thermodynamic Calculations taking into account base stacking energyThe nearest neighbor and thermodynamic calculations are done essentially as described by Breslauer et al., (1986) Proc. Nat. Acad. Sci. 83:3746-50 (Abstract) but using the values published by Sugimoto et al., (1996) Nucl. Acids Res. 24:4501-4505 (Abstract). RNA thermodynamic properties were taken from Xia T., SantaLucia J., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., Turner D.H. (1998) Biochemistry 37:14719-14735 (Abstract). This program assumes that the sequences are not symmetric and contain at least one G or C. The minimum length for the query sequence is 8.
The melting temperature calculations are based on the thermodynamic relationship between entropy, enthalpy, free energy and temperature, where
The thermodynamic calculations assume that the annealing occurs at pH 7.0. The melting temperature (Tm) calculations assume the sequences are not symmetric and contain at least one G or C. The oligonucleotide sequence should be at least 8 bases long to give reasonable Tms. The accuracy of the calculation decreases after 20 nucleotides since the equations and parameters were defined with oligonucleotides in the size range of 14-20 nucleotides. Monovalent cations concentrations (either Na+ or K+) should be between 0.01 and 1.0 M. None of the following melting temperature (Tm) calculations provide an adjustment for magnesium or manganese divalent cation concentration, although lack of these cations has been shown to adversely affect proper duplex formation. See Nakano et al, (1999) Nucleic Acids Res 27:2957-65. (Abstract)
Basic Melting Temperature (Tm) CalculationsThe two standard approximation calculations are used. For sequences less than 14 nucleotides the formula is
Tm= (wA+xT) * 2 + (yG+zC) * 4
where w,x,y,z are the number of the bases A,T,G,C in the sequence,
respectively (from Marmur,J., and Doty,P. (1962) J Mol Biol 5:109-118 [PubMed]).
Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)
See Wallace,R.B., Shaffer,J., Murphy,R.F., Bonner,J., Hirose,T., and Itakura,K. (1979) Nucleic Acids Res 6:3543-3557 (Abstract) and Sambrook,J., and Russell,D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY. (CHSL Press)
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.
Salt Adjusted Melting Temperature (Tm) CalculationsA variation on two standard approximation calculations are used. For sequences less than 14 nucleotides the same formula as the basic calculation is use, with a salt concentration adjustment
Tm= (wA+xT)*2 + (yG+zC)*4 - 16.6*log10(0.050) +
where w,x,y,z are the number of the bases A,T,G,C in the sequence,
For sequences longer than 13 nucleotides, the equation used is
Tm= 100.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (820/(wA+xT+yG+zC)) +
The following equation is provided only for your reference. It is not actually used by OligoCalc. It is reportedly more accurate for longer sequences.
Tm= 81.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (500/(wA+xT+yG+zC)) +
16.6*log10([Na+]) - 0.62F
For more information please see the reference:
Howley, P.M; Israel, M.F.; Law, M-F.; and M.A. Martin "A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes." J. Biol. Chem. 254, 4876-4883, 1979.
RNA melting temperatures
Tm= 79.8 + 18.5*log10([Na+]) + (58.4 * (yG+zC)/(wA+xT+yG+zC))
+ (11.8 * ((yG+zC)/(wA+xT+yG+zC))2) - (820/(wA+xT+yG+zC))
These equations assume that the annealing occurs under the standard conditions of 50 nM primer and pH 7.0.
Melting Temperature Method Comparisons
The Basic Melting Temperature calculations are provided as a baseline for comparison, and are the least preferred, however are perhaps the most often employed method for calculating melting temperature by bench scientists. OligoCalc was designed to give researchers an easy tool for finding and comparing melting temperatures using more accurate calculations. For oligonucleotides between 8 and 40 nucleotides, the nearest neighbor method is the preferred method. Note that the equations were developed using 14-20mers, so this method is the most accurate for oligonucleotides of this length. A comparison of these data sets and recommendations were recently published (Panjkovich,A. and Melo,F. (2005) Bioinformatics 21:711-722 [abstract]) and implemented as a webserver (Panjkovich,A., Norambuena,T. and Melo,F. (2005) dnaMATE: a consensus melting temperature prediction server for short DNA sequences. Nucleic Acids Res 33:W570-W572. [abstract]), and predominantly agree with the methods we have chosen. For longer sequences, or for oligonucleotides with base substitutions or modifications, the Salt Adjusted Melting Temperature calculation is the preferred method. Please note that these calculations are only estimates and many other factors can affect the melting temperature, including detergents, presence of other counter ions, solvents (ethanol for instance), formamide, etc.
Molecular Weight Calculations
DNA Molecular Weight (typically for synthesized DNA oligonucleotides. The OligoCalc DNA MW calculations assume that there is not a 5' monophosphate)
Anhydrous Molecular Weight = (An x 313.21) + (Tn x 304.2) + (Cn x 289.18) + (Gn x 329.21) - 61.96
An, Tn, Cn, and Gn are the number of each respective nucleotide within the polynucleotide. The subtraction of 61.96 gm/mole from the oligonucleotide molecular weight takes into account the removal of HPO2 (63.98) and the addition of two hydrogens (2.02). Alternatively, you could think of this of the removal of a phosphate and the addition of a hydroxyl, since this formula calculates the molecular weight of 5' and 3' hydroxylated oligonucleotides.
Please note: this calculation works well for synthesized oligonucleotides. If you would like an accurate MW for restriction enzyme cut DNA, please use:
Molecular Weight = (An x 313.21) + (Tn x 304.2) + (Cn x 289.18) + (Gn x 329.21) - 61.96 + 79.0
The addition of 79.0 gm/mole to the oligonucleotide molecular weight takes into account the 5' monophosphate left by most restriction enzymes. No phosphate is present at the 5' end of strands made by primer extension, so no adjustment to the OligoCalc DNA MW calculation is necessary for primer extensions. That means that for ssDNA, you need to add 79.0 to the value calculated by OligoCalc to get the weight with a 5' monophosphate. Finally, if you need to calculate the molecular weight of phosphorylated dsDNA, don't forget to adjust both strands. You can automatically perform either addition by selecting the Phosphorylated option from the 5' modification select list. Please note that the chemical modifications are only valid for DNA and may not be valid for RNA due to differences in the linkage chemistry, and also due to the lack of the 5' phosphates from synthetic RNA molecules.
RNA Molecular Weight (for instance from an RNA transcript. The OligoCalc RNA MW calculations assume that there is a 5' triphosphate on the molecule)
Molecular Weight = (An x 329.21) + (Un x 306.17) + (Cn x 305.18) + (Gn x 345.21) + 159.0
An, Un, Cn, and Gn are the number of each respective nucleotide within the polynucleotide. Addition of 159.0 gm/mole to the molecular weight takes into account the 5' triphosphate.
Clicking on the BLAST button will perform a BLAST search at the NCBI against the NR database. For more information about BLAST, please go to the main BLAST server site and read about the many, many options available there.
- About BLAST: http://www.ncbi.nlm.nih.gov/blast/producttable.html
- BLAST main site: http://www.ncbi.nlm.nih.gov/blast/
- BLAST FAQs: http://www.ncbi.nlm.nih.gov/blast/blast_FAQs.html
Please go to the main mfold webserver for more information about mfold and the many, many excellent features and options it has for determining folding structures for DNA and RNA. mfold was written by Professor Michael Zuker and colleagues at RPI. See Zuker,M. (2003) "Mfold web server for nucleic acid folding and hybridization prediction" Nucleic Acids Res 31:3406-15 (abstract) and Mathews,D.H., Sabina,J., Zuker,M. and Turner, D.H. (1999) J Mol Biol 288:911-940 (abstract).
- mfold documentation: http://www.bioinfo.rpi.edu/applications/mfold/doc/form1-doc.html
- mfold main site: http://www.bioinfo.rpi.edu/applications/mfold/
Professor Zuker has also released a more advanced folding application, and that package is available here.
Self-dimerization, hairping formation, and self-annealing of 3' and 5' ends
These methods have been replaced by the mfold webserver linkout, but for those who want to compare the methods, I am providing the old method as well. Clicking the 'Check Self-Complementarity' button results in a new window with likely hairpin and self-complementary areas highlighted. The structures shown are based solely on homology and length of homology as well as some rudimentary constraints for ends, size of hairpins, etc. These calculations were based on available models and assumptions as described in:
- Hilbers,C.W., Blommers,M.J.J., Haasnoot,C.A.G., van der Marel, G.A. and van Boom, J. H. (1987) Structure and folding of DNA and RNA hairpins. Anal Chem 327:70 (not in PubMed)
- Serra,M.J., Lyttle,M.H., Axenson,T.J., Schadt.C.A. and Turner,D.H. (1993) Nucleic Acids Res 21:3845-3849 (abstract) and Vallone,P.M., Paner,T.M., Hilario,J., Lane,M.J., Faldasz,B.D., Benight,A.S. (1999) Biopolymers. 50, 425-442 (abstract)
The calculations are also available as part of the OligoCalc source code.
Molar Absorptivity values in 1/(Moles cm)
|Residue||Moles-1 cm-1||Amax(nm)||Molecular Weight
(after protecting groups are removed)
|Adenine (dAMP, Na salt)||15200||259||313.21|
|Guanine (dGMP, Na salt)||12010||253||329.21|
|Cytosine (dCMP, Na salt)||7050||271||289.18|
|Thymidine (dTMP, Na salt)||8400||267||304.2|
|dUradine (dUMP, Na salt)||9800||-||290.169|
|dInosine (dUMP, Na salt)||-||-||314|
|Adenine (AMP, Na salt)||15400||259||329.21|
|Guanine (GMP, Na salt)||13700||253||345.21|
|Cytosine (CMP, Na salt)||9000||271||305.18|
|Uradine (UMP, Na salt)||10000||262||306.2|
Assume 1 OD of a standard 1ml solution, measured in a cuvette with a 1 cm pathlength.
|Absorption wavelength maximum:||495 nm|
|Emission wavelength maximum:||521 nm|
|Molar Absorptivity at 260nm:||20960 Moles-1 cm-1|
|Chemical name:||4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein|
|Absorption wavelength maximum:||519 nm|
|Emission wavelength maximum:||539 nm|
|Molar Absorptivity at 260nm:||16255 Moles-1 cm-1|
|Chemical name:||4, 7, 2', 4', 5', 7'-Hexachloro-6-carboxyfluorescein|
|Absorption wavelength maximum:||537 nm|
|Emission wavelength maximum:||556 nm|
|Molar Absorptivity at 260nm:||31580 Moles-1 cm-1|
|Chemical name:||N, N, N', N'-tetramethyl-6-carboxyrhodamine|
|Absorption wavelength maximum:||555 nm|
|Emission wavelength maximum:||580 nm|
|Molar Absorptivity at 260nm:||31980 Moles-1 cm-1|
Nucleotide base codes (IUPAC)
Most recent version is available at URL: http://www.basic.northwestern.edu/biotools/oligocalc.html
If you have comments, kudos, raspberries, please contact:
Warren A. Kibbe, Ph.D.
Robert H. Lurie Comprehensive Cancer Center
Feinberg School of Medicine
Chicago, IL 60611
Original code by Eugen Buehler
Research Support Facilities
Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine
Hairpin and complementarity checking code by Qing Cao, M.S.
While working in Research Computing (1999-2001)
Northwestern University Medical School
Chicago, IL 60611
Monomer structures and molecular weights provided by Bob Somers, Ph.D. at Glen Research Corporation
Uppercase/lowercase strand complementation problem described by Alexey Merz
The RNA calculations and functions were requested by Suzanne Kennedy, Ph.D. at Qiagen
The fluorescent tags and tagging options were requested by and the data provided by Florian Preitauer and Regina Bichlmaier, Ph.D. at metabion GmbH
The dsDNA and dsRNA molecular weight calculation problems in version 3.xx and fixed in 3.10 was reported by Borries Demeler, Ph.D. and Regina Bichlmaier, Ph.D. at metabion GmbH