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INTEGRATEDDNATECHNOLOGIESPRODUCTSAPPENDIXMolar Extinction CoefficientOptical absorbance at 260 nm is routinely used to measure the concentration of nucleic acids present in a solution. Approximate conversionfactors estimate that duplex DNA is about 50 µg/OD 260, single-stranded RNA is approximately 40 µg/OD 260, and single-stranded DNA isapproximately 33 µg/OD 260. While this is true for randomized sequences, these conversion factors are less accurate for short oligonucleotidesand repeating sequences. Since the absorbance of each base is different, base composition and sequence context influence the absorbance.For example, 1.0 OD 260of d(CCCCCCCCCCCC) (homopolymeric deoxycytidine) has a mass of 39 µg while 1.0 OD 260of d(AAAAAAAAAAAA)(homopolymeric deoxyadenosine) has a mass of 25 µg. The extinction coefficient (ε 260) describes the relationship between concentrationand UV absorbance and can be calculated for any sequence. Greatest accuracy is therefore achieved when the exact value of ε 260iscalculated for each oligo. Further, it is possible to take into account the presence of modified groups, such as fluorescent dyes, which havesignificant 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(A 260) of 1 mole/L DNA solution measured in 1 cm path-length cuvette. This definition is derived from the Beer-Lambert law,A = log(I O/ I) = ε * c * pwhere A is the absorbance, I Oand I are, respectively, the intensities of incident and transmitted light, c is the molar concentration of anoligonucleotide (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 ε 260value 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),ε 260= 2x (Σε Nearest-Neighbor ) - Σε Individual + Σε ModificationN-1 N-1 N1 2 1where ε Nearest-Neighboris the nearest neighbor coefficient for a pair of bases, ε Individualis the coefficient for an individual base, and N is the lengthof the oligonucleotide.Molecular Weight (Anhydrous)Molecular weight (MW) is the sum of the atomic masses of the constituent atoms for 1 nmole of oligonucleotide. The anhydrous molecularweight represents the pure oligo free of any of the counter ions or water molecules that are normally weakly bound to an oligo aftersynthesis. This calculation gives the molecular weight as measured by mass spectrometry.Molecular weight of an oligomer is a sum of the weights of individual bases and chemical modifications. Oligos are typically synthesizedwithout a 5’-phosphate group, which must be subtracted.where PO 2H = 63.980 and H 2= 2.016Anhydrous MW = Σ Individual Base MW+ Σ Individual Mod MW- PO 2H + H 2Molecular weights of DNA bases:dA 313.2dC 289.2dG 329.2dT 304.2dU 290.2dI 314.2RNA bases: The molecular weight of an RNA nucleotide is the weight of a DNA nucleotide + 15.999, accounting for the additional oxygenatom present (Example: rA is dA (313.209) + 15.999 = 329.208). When determining the weight of uracil (rU), start with dU and not thymine(dT).2’-O-Methyl bases: The molecular weight of a 2’-O-Methyl RNA nucleotide is the weight of a DNA nucleotide + 30.026, accounting for theadditional methoxy group (-OCH 3) present (For example: mA is dA (313.209) + 30.026 = 343.235). When determining the weight of uracil(mU), start with dU and not thymine (dT).©2008 Integrated DNA Technologies www.idtdna.com91
INTEGRATEDDNATECHNOLOGIESPRODUCTSAPPENDIXMolecular Weight (Anhydrous) continuedLNA bases: The molecular weight of an LNA nucleotide is the weight of a DNA nucleotide + 28.011, accounting for the bridging oxygen andcarbon from the 2’ carbon to the 4’ carbon (-OCH 2-) present (For example: +A is dA (313.209) + 28.011 = 341.220). The exception is LNA C,which contains an additional methyl group off of the 5-carbon (14.026).Phosphorothioated bases: The molecular weight of a phosphorothioate nucleotide is the weight of a nucleotide (DNA, RNA, 2’-O-Methyl,LNA) + 16.061, accounting for the substitution of one sulfur atom for a non-bridging oxygen atom in the phosphodiester backbone (Forexample: A* is A (313.209) + 16.061 = 329.270). Phosphorothioate modification refers to substitutions affecting the internucleoside linkagesand does not involve the free 3’- or 5’- ends. Thus a 20-mer phosphorothioate oligonucleotide has 19 phosphorothioate linkages.nmole/OD 260The amount of oligonucleotide in nanomoles that, when dissolved in 1 mL volume, results in 1 unit of absorbance at 260 nm with a standard1 cm path-length cuvette. nmole/OD 260is calculated from an oligonucleotide’s molar extinction coefficient. OD 260is calculated from thefollowing equation,OD 260= (A 260* V) / pwhere A 260is the absorbance at 260 nm, V is the solution volume in mL, and p is the length of the light path through the sample (cm). Thus,OD 260has the units mL/cm. Starting from the oligo molar extinction coefficient, ε 260,ε 260= L/(mol . cm) = 10 3 mL/(mol . cm)Since OD 260has the units of mL/cm, the equation can be written as,10 3 OD 260/mol = 10 3 OD 260/ 10 9 nmol = OD 260/ 10 -6 nmolCombination of both equations yields,ε 260= OD 260/ 10 -6 nmoland this can be rearranged as,nmole/OD 260= 10 6 / ε 260µg/OD 260The amount of oligonucleotide in micrograms that, when dissolved in 1 mL volume, results in 1 unit of absorbance at 260 nm with astandard 1 cm path-length cuvette. µg/OD 260is derived using molecular weight and nmole/OD 260values:µg/OD 260= nmole/OD 260* molecular weight (g/mol) * 10 -3The definition of OD 260can be found within the nmole/OD 260definition above.©2008 Integrated DNA Technologies 92 www.idtdna.com
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