Optical absorbance at 260 nm is routinely used to measure the concentration of nucleic acids present in a solution. Approximate conversion factors estimate that duplex DNA is about 50 µg/OD260, single-stranded RNA is approximately 40 µg/OD260, and single-stranded DNA is approximately 33µg/OD260. While this is true for randomized sequences, these conversion factors are less accurate for short oligonucleotides and repeating sequences. Since the absorbance of each base is different, base composition and sequence context influence the absorbance. For example, 1.0 OD260 of d(CCCCCCCCCCCC) (homopolymeric deoxycytidine) has a mass of 39 µg while 1.0 OD260 of d(AAAAAAAAAAAA) (homopolymeric deoxyadenosine) has a mass of 25 µg. The extinction coefficient (ε260) describes the relationship between concentration and UV absorbance and can be calculated for any sequence. Greatest accuracy is therefore achieved when the exact value of ε260 is calculated for each oligo. Further, it is possible to take into account the presence of modified groups, such as fluorescent dyes, which have significant absorbance at 260 nm.
The molar extinction coefficient is a physical constant that is unique for each sequence and describes the amount of absorbance at 260 nm (A260) of 1 mole/L DNA solution measured in 1 cm path-length cuvette. This definition is derived from the Beer-Lambert law,
A = log(IO/I) = ε * c * p
where A is the absorbance, IO and I are, respectively, the intensities of incident and transmitted light, c is the molar concentration of an oligonucleotide(mole/L), p is the length of the light path through the sample (cm), and ε is the molecule molar extinction coefficient (L/(mole ·cm)). The ε260 value of an oligonucleotide is calculated from the following equation (Cantor,C.R. et al., Biopolymers, 9, 1059-1077., Cavaluzzi,M.J. and Borer,P.N. Nucleic Acids Res., 32, e13),
where εNearest Neighbor is the nearest neighbor coefficient for a pair of bases, εIndividual is the coefficient for an individual base, and N is the length of the oligonucleotide.