A semi-micromethod for the determination of the extinction coefficients of duplex and single-stranded DNA

We have developed a rapid and convenient procedure for the determination of concentrations and extinction coefficients of oligo- and polynucleotides. It offers significant advantages over other methods in terms of precision and the ability to detect artifactual or erroneous results. Samples are first completely digested with appropriate enzymes to mononucleotides and nucleosides. Using the multicomponent linear regression capabilities of commonly available spreadsheet programs, the absorbance spectrum of the digest can be analyzed as a linear combination of the contribution of the possible constituent monomers. If all the spectral components present have been included, the analysis yields the concentration of each of the monomer species whose sum is the concentration (in monomer units) of the original undigested sample. When combined with the predigest absorbance spectrum, the extinction coefficients of the intact sample can then be calculated. The analysis also yields the fractional base composition of the oligomer or polymer. The extensive spectral data provided by digital read-outs of modern spectrophotometers permit the application of sensitive tests of the goodness of fit, thus facilitating the detection of artifacts and sample inhomogeneity. Both single-strand and duplex structures can be analyzed comfortably in sample sizes of 25 to 35 nmol (total) of mononucleotides with a precision of 1%. The concentrations obtained by this method agree, on the average, within 0.2% with those determined by phosphate analysis of the same sample. The method also yields the base composition with an accuracy of ca. 5% for high-molecular-weight polymers and 2% for short oligomers (15-20 bp) when compared to the predicted values.