Oligonucleotide Properties Calculator

Enter Oligonucleotide Sequence Below
OD and Molecular Weight calculations are for single-stranded DNA
Nucleotide base codes

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

Number of Fluorescent tags per strand:
6-FAM  TET   HEX   TAMRA
Minimum base pairs required for single primer self-dimerization:
Minimum base pairs required for a hairpin :
Physical Constants
Melting Temperature (TM) Calculations
Length: bases
GC content: %
Molecular Weight:
1 ml of a sol'n with an Absorbance of at 260 nm
is microMolar 4 and contains micrograms.
 1 °C (Basic)
2 °C (Salt Adjusted)
3 °C (Nearest Neighbor)
nM Primer
mM Salt (Na+)
Thermodynamic Constants
Conditions: 1 M NaCl at 25°C at pH 7.
RlogK 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 is also 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, 1997-2002.

About the Calculations

Thermodynamic Calculations

The nearest neighbor and thermodynamic calculations are done essentially as described by Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50, 1986 (Abstract) but using the values published by Sugimoto et al., Nucl. Acids Res. 24, 4501-4505, 1996 (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

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 (820/(wA + xT + yG + zC)) adjusting for the length of the sequence.

ASSUMPTIONS:
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer and pH 7.0.

OD Calculations

Molar Absorptivity values in 1/(Moles cm)

Residue Moles-1 cm-1 Molecular Weight
(after protecting groups are removed)
Adenine (dAMP, Na salt) 15200 313.21
Guanine (dGMP, Na salt) 12010 329.21
Cytosine (dCMP, Na salt) 7050 289.18
Thymidine (dTMP, Na salt) 8400 304.2
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
    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. e-mail
Research Computing
Northwestern University Medical School
Chicago, IL 60611

Warren A. Kibbe, Ph.D. e-mail 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.
Sr. Applications Chemist
Glen Research Corporation
22825 Davis Drive
Sterling, VA 20164
http://www.glenres.com/

Uppercase/lowercase strand complementation problem described by Alexey Merz alexey@dartmouth.edu