Oligo Calc: Oligonucleotide Properties Calculator

Enter Oligonucleotide Sequence Below
OD calculations are for single-stranded DNA or RNA
Nucleotide base codes

Reverse Complement Strand(5' to 3') is:

5' modification (if any) 3' modification (if any) Select molecule
        nM Primer         Measured Absorbance at 260 nanometers
        mM Salt (Na+)
Physical Constants
Melting Temperature (TM) Calculations
Length: Molecular Weight: 4 GC content: %
1 ml of a sol'n with an Absorbance of at 260 nm
is microMolar 5 and contains micrograms.
1 °C (Basic)
2 °C (Salt Adjusted)
3 °C (Nearest Neighbor)
Thermodynamic Constants Conditions: 1 M NaCl at 25°C at pH 7.
RlnK cal/(°K*mol) deltaH Kcal/mol
deltaG Kcal/mol deltaS cal/(°K*mol)
Deprecated Hairpin/self dimerization calculations
    (Minimum base pairs required for single primer self-dimerization)
    (Minimum base pairs required for a hairpin)
Citation: Kibbe WA. 'OligoCalc: an online oligonucleotide properties calculator'. (2007)
    Nucleic Acids Res. 35(webserver issue): May 25. ( Abstract/Full text)
This page may be freely linked or distributed for any educational or non-commercial use.
Copyright (c) Northwestern University, 1997-2010.
Localized versions of OligoCalc are available in Russian.
Version history
Web traffic statistics for 2006
OligoCalc Usage Patterns
Table of chemical modifications and structures
Source Code
Russian language version

 


Melting Temperature (Tm) Calculations

ASSUMPTIONS:
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)

Thermodynamic Calculations taking into account base stacking energy

The 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 change in entropy (order or a measure of the randomness of the oligonucleotide) and enthalpy (heat released or absorbed by the oligonucleotide) are directly calculated by summing the values for nucleotide pairs obtained by Sugimoto et al., (1996) Nucleic Acids Res 24:4501-4505 (Abstract). The relationship between the free energy and the concentration of reactants and products at equilibrium is given by

where [DNA•primer] is the concentration of the bound DNA•primer complex, [DNA] is the concentration of unbound DNA target sequence, and [primer] is the concentration of unbound primer. Substituting for ΔG, the two equations give us:

and solving for temperature T gives

We can assume that the concentration of DNA and the concentration of the DNA-primer complex are equal (that is, the concentration of primer is in excess of the target DNA and the melting point is where the concentration of bound and unbound DNA are at equilibrium), so this simplifies the equation considerably. If the two strands are in equal concentration, the effective concentration is 0.25 the total concentration of oligonucleotide (Wetmur,J.G., (1991) Crit Rev Biochem Mol Biol 26:227-259 [Abstract). It has been determined empirically that there is a 5 kcal free energy change (3.4 by Sugimoto et al. and the value used by OligoCalc) during the transition from single stranded to B-form DNA. This represents the helix initiation energy. Finally, adding an adjustment for salt gives the equation that the OligoCalc uses:

An adjustment constant for salt concentration is not needed, since the various parameters were determined at 1 Molar NaCl, and the log of 1 is zero.

ASSUMPTIONS:
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) Calculations

The two standard approximation calculations are used. For sequences less than 14 nucleotides the formula is

For sequences longer than 13 nucleotides, the equation used is

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)

ASSUMPTIONS:
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) Calculations

A 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

The term 16.6*log10([Na+]) adjusts the Tm for changes in the salt concentration, and the term log10(0.050) adjusts for the salt adjustment at 50 mM Na+. Other monovalent and divalent salts will have an effect on the Tm of the oligonucleotide, but sodium ions are much more effective at forming salt bridges between DNA strands and therefore have the greatest effect in stabilizing double-stranded DNA, although trace amounts of divalent cations have significant and often overlooked affects (See Nakano et al, (1999) Proc. Nuclec Acids Res. 27:2957-65. (Abstract)).
For sequences longer than 13 nucleotides, the equation used is

This equation is accurate for sequences in the 18-25mer range (Howley,P.M., Israel,M.F., Law,M-F., and Martin,M.A. (1979) J Biol Chem 254:4876-4883 [Abstract]). OligoCalc uses the above equation for all sequences longer than 13 nucleotides.

The following equation is provided only for your reference. It is not actually used by OligoCalc. It is reportedly more accurate for longer sequences.

This equation is most accurate for sequences longer than 50 nucleotides. It is valid for oligos longer than 50 nucleotides from pH 5 to 9. Symbols and salt adjustment term as above, with the term (41 * (yG + zC-16.4)/(wA + xT + yG + zC)) adjusting for G/C content and the term (500/(wA + xT + yG + zC)) adjusting for the length of the sequence, and F is the percent concentration of formamide.

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

Where yG+zC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex (From Sambrook,J., and Russell,D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY. (CHSL Press).
ASSUMPTIONS:
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.


BLAST linkout

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.


mfold linkout

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).

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:

The calculations are also available as part of the OligoCalc source code.


Optical Density (OD) Calculations

Molar Absorptivity values in 1/(Moles cm)

ResidueMoles-1 cm-1Amax(nm)Molecular Weight
(after protecting groups are removed)
Adenine (dAMP, Na salt) 15200259313.21
Guanine (dGMP, Na salt) 12010253329.21
Cytosine (dCMP, Na salt) 7050271289.18
Thymidine (dTMP, Na salt) 8400267304.2
dUradine (dUMP, Na salt) 9800-290.169
dInosine (dUMP, Na salt) --314
RNA nucleotides
Adenine (AMP, Na salt) 15400259329.21
Guanine (GMP, Na salt) 13700253345.21
Cytosine (CMP, Na salt) 9000271305.18
Uradine (UMP, Na salt) 10000262306.2
Other nucleotides
6' FAM 20960 537.46
TET 16255 675.24
HEX 31580 744.13
TAMRA 31980

Assume 1 OD of a standard 1ml solution, measured in a cuvette with a 1 cm pathlength.


6-FAM:

Chemical name: 6-carboxyfluorescein
Absorption wavelength maximum: 495 nm
Emission wavelength maximum: 521 nm
Molar Absorptivity at 260nm: 20960 Moles-1 cm-1


TET:

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


HEX:

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


TAMRA:

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)

Symbol: nucleotide(s)
    A adenine
    C cytosine
    G guanine
    T thymine in DNA;
    uracil in RNA
    U deoxy-Uracil in DNA;
    uracil in RNA
    I inosine
    N A or C or G or T
M A or C
R A or G
W A or T
S C or G
Y C or T
K G or T
V A or C or G; not T
H A or C or T; not G
D A or G or T; not C
B C or G or T; not A

The most recent version is always available at the URL: http://www.basic.northwestern.edu/biotools/OligoCalc.html


OligoCalc was written in Javascript.

 

If you have comments, kudos, raspberries, please contact:

 

Original code (1996) by Eugen Buehler

 

Hairpin and complementarity checking code by Qing Cao, M.S.

 

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

 

A failure to include 'U's in the OD calculations was reported by Rachel Mitton-Fry at Yale University

 

The Russian localization of OligoCalc was done by Leonid Valentovich

 

A longstanding failure to include A260s for 3' and 5' modifications in the OD calculations was reported by Kate Lieberman at the University of California, Santa Cruz.

 

An increase in precision to 0.01 degree increments for SNP analysis and HRM was requested by Zhao Chen at the MD Anderson Cancer Center.