Concentration and Extinction Coefficient Determination for Oligonucleotides and Analogs Using a General Phosphate Analysis

Abstract

A general phosphate analysis (GPA) is developed which assays the concentration of nucleic acid oligomers and their analogs based on stoichiometric phosphorus in the sequence. The method involves complete digestion of the oligomer sample to orthophosphate using acid at high temperature and subsequent colorimetric analysis by phosphomolybdate complex formation. GPA is applied to oligomers having phosphodiester, methylphosphonate, and phosphorothioate backbone linkages. Given the absorption spectra of oligomers having these backbones, extinction coefficients are obtained and compared to other quantitative and predictive methods. In addition to sequences having the usual nucleoside residues found in naturally occurring nucleic acids, oligomers having base analog residues can be readily quantified by GPA.