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  orderingAn Introduction to Extinction Coefficients and Molecular Weights of Oligonucleotides
By Judy Ngo and Shawna Oliva; TriLink BioTechnologies

Every TriLink Certificate of Analysis lists two numbers crucial to the use of an oligonucleotide: the extinction coefficient and the molecular weight. These numbers are needed to calculate important data, such as determining molar concentrations and preparing stock solutions. This paper will offer an explanation of the extinction coefficients and molecular weights of oligonucleotides, discuss the means by which the numbers are derived, and give practical examples on how to use the data provided by TriLink.

The optical density unit, or more commonly the OD260 unit, is a spectrophotometric measurement of an oligonucleotide. Each of the bases in a nucleic acid strand has an absorbance at or near 260 nanometers, due to their conjugated double bond systems. Because the exact base sequence and composition is known, the OD260 unit is a very accurate and convenient method to quantify an oligonucleotide. The OD260 unit is a normalized unit of measurement that is defined as the amount of oligonucleotide required to give an absorbance reading of 1.0 at 260 nanometers in 1.0 milliliter of solution using a 1.0 centimeter light path. Utilizing absorbance measurements is the recommended method for quantitating or aliquoting an oligonucleotide. Often, the total mass of oligonucleotide is too low to accurately weigh on a balance. In addition, spectrophotometric measurements will be accurate regardless of any excess salt present in the sample. An absorbance reading will also ensure that a compound is fully solubilized prior to use.

Associating the OD260 unit with the amount of oligo present is done mathematically using a physical constant known as the extinction coefficient. It is a component of Beer's Law: A = εCl; where A is absorbance, C is concentration, l is the path length, and ε is the extinction coefficient, a constant for the material being analyzed. The extinction coefficient takes into account the effects of the neighboring bases as well as the absorbance of each of the individual bases. Because it is dependent on the exact nucleotide composition and sequence, the extinction coefficient is unique to every oligonucleotide. There are several ways to determine the extinction coefficient of an oligonucleotide. The "nearest neighbor" method gives greater accuracy than the more common method of merely adding the individual bases and multiplying by the individual extinction coefficients. TriLink uses this "nearest neighbor" model to calculate the extinction coefficient of each oligonucleotide. The next page shows the formula for calculating the extinction coefficient as well as the established constants for both the DNA and RNA nucleotides and dimer pairs. TriLink's website conveniently offers a spreadsheet that automatically calculates the extinction coefficient of an oligonucleotide.

The extinction coefficient is listed on TriLink's Certificate of Analysis and is expressed in ODs/µmole. These units are mathematically derived and equivalent to the standard equation units, L mmole-1 cm-1. Please note that although TriLink uses the relatively accurate "nearest neighbor" model to calculate the extinction coefficient, it is still just an approximation and may be off by as much as 10%.

Another important piece of data found on TriLink's Certificate of Analysis is the molecular weight. The molecular weight of an oligonucleotide is simply the mass of the compound in grams per mole (6.02 x 1023 molecules). It is the sum of each of the component molecular weights of all the atoms the oligonucleotide may contain. This value is needed for converting OD260 units into units of mass. TriLink gives the molecular weight of the free acid form of the oligonucleotide. The next page shows the information used to calculate the molecular weight of an unmodified oligonucleotide.

Since TriLink provides the final yield of the product in OD260 units, it is important to understand how to use this data to convert from OD260 units into other desired units. Below are examples of conversions to some of the more commonly used units. If you have any further questions, please contact our Client Relations Department. We will be happy to assist you.

Oligo Unit Conversion

Sample sequence:
5' AGC TAA GTC ACT GCC ATT GA 3'

Extinction Coefficient (ε)*: 195.5 OD units/µmole
Molecular Weight (MW): 6101.0 g/mole
OD (A260): 32.4

Example 1: Converting to µmoles

µmoles of oligo = OD (A260) ÷ ε
= 32.4 ÷ 195.5 OD units/µmole
= 0.166 µmoles

Example 2: Converting to µgrams
(Use the µmoles calculated in Example 1)

µgrams of oligo = µmoles x MW
= 0.166 µmoles x 6101.0 g/mole
= 1012.8 µgrams

*If you are working in L mmole-1cm-1 units, you will first need to convert the ε to OD units/µmole.
ε L mmole-1cm-1 ÷ 1000 = ε OD units/µmole
195,500 L mmole-1cm-1 ÷ 1000 = 195.5 OD units/µmole

Oligonucleotide Extinction Coefficient Formula

ε 260
 
ε 260
pdA 15.4 dCpdG 9.0
pdC 7.4 dCpT 7.6
pdG 11.5 dGpdA 12.6
pT 8.7 dGpdC 8.8
dApdA 13.7 dGpdG 10.8
dApdc 10.6 dGpT 10.0
dApdG 12.5 TpdA 11.7
dApT 11.4 TpdC 8.1
dCdpA 10.6 TpdG 9.5
dCpdC 7.3 TpT 8.4

To calculate the number of units OD260 per µmole of oligonucleotide with the sequence 5' DpEp...KpL, use the following formula with the table above:
ε 260 DpEpFpGp...KpL = [2(ε DpE + ε EpF + ε FpG + .. + ε KpL) - ε E - ε F - ε G - .. - ε K ]

Note that you do not include the 3' and 5' terminal bases in the second part (monomers) of the equation, whereas you use every dimer par in the first part.

Oligonucleotide Molecular Weight Calculations

To calculate the molecular weight of an oligonucleotide, use the formula below.

  (# of dA in sequence) x 249.24
  + (# of dC in sequence) x 225.23
  + (# of dG in sequence) x 265.2
  + (# of T in sequence) x 240.23
  + (# of phosphodiester linkages in sequence) x 63.97
  + (# of phosphorothioate linkages in sequence) x 80.08
  + 2 for 3' and 5' terminal hydrogens    
 
= Molecular weight of oligonucleotide
   
 

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