Oligonucleotide Properties Calculator

Enter Oligonucleotide Sequence Below
OD and Molecular Weight 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 ss/ds and DNA or RNA
        (Minimum base pairs required for single primer self-dimerization)
        (Minimum base pairs required for a hairpin)
        nM Primer
        mM Salt (Na+)
Physical Constants
Melting Temperature (TM) Calculations
Length: bases
GC content: %
Molecular Weight: 4
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)
To use this calculator, you must be using Netscape 3.0 or later
or Internet Explorer version 3.0 or later, or another Javascript-capable browser
Self-Complementarity requires a 4.x browser. IE 5.0, Safari, and Mozilla supported.
This page was written in Javascript.
Extensively rewritten from 12/15/2000-12/19/2000 to isolate javascript Oligo object behaviors for teaching purposes.
This page may be freely distributed for any educational or non-commercial use.
Copyright Northwestern University, 1997-2006.
Version history

About the Calculations

Thermodynamic Calculations

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 simple 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 Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50, 1986. The relationship between the free energy and the concentration of reactants and products at equilibrium is given by

Substituting the two equations gives 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, so this simplifies the equation considerably. It has been determined empirically that there is a 5 (3.4 by Sugimoto et al.) kcal free energy change during the transition from single stranded to B-form DNA. This is presumably a helix initiation energy. Finally, adding an adjustment for salt gives the equation that the Oligo Calculator uses:

No adjustment constant for salt concentration is 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.

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

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.
For sequences longer than 13 nucleotides, the equation used is

This equation is very accurate for sequences in the 18-25mer range. 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.
ASSUMPTIONS:
These equations assume that the annealing occurs under the standard conditions of 50 nM primer and pH 7.0.

Molecular Weight Calculations

DNA Molecular Weight (for instance synthesized Oligonucleotides)

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).

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) + 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 should be necessary. Also, if you are making these calculations for dsDNA, the correct amount to add it 158.0 gm/mole, adding a monophosphate to each strand.

RNA Molecular Weight (for instance from an RNA transcript)
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.

OD Calculations

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
RNA nucleotides
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
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

Most recent version is available at URL: http://www.basic.northwestern.edu/biotools/oligocalc.html

The current version is the result of efforts by the following people:

Qing Cao, M.S.
Research Computing
Northwestern University Medical School
Chicago, IL 60611

Warren A. Kibbe, Ph.D. and PH entry.
Research Computing
Northwestern University Medical School
Chicago, IL 60611

Original code by Eugen Buehler
Research Support Facilities
Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine

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 flourescent 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