An improved method for the determination of oligonucleotide chain length using phosphodiesterase hydrolysis and ion-pair high-performance liquid chromatography

An improved method for the determination of chain length, leading and/or terminating nucleotide, and base composition of the deoxyribo- and ribooligonucleotides was developed, based upon the phosphodiesterase-catalyzed hydrolysis of the oligonucleotide, followed by ion-pair (hetaeric) high-performance liquid chromatography using tetrabutyl ammonium phosphate as the hetaeron. Chains containing terminal phosphate groups were first dephosphorylated using alkaline phosphatase. Total analysis times involved a 1.5-h phosphodiesterase incubation, followed by a 40-min chromatographic separation. Minimal or no sample preparation is involved. An analysis of the propagation of errors indicated that chains of up to 36 nucleotides in length could be analyzed with less than a 0.5-base-unit error. Ultimate sensitivity will depend upon the particular high-performance liquid chromatography system, and will be limited by the detection limits for a single nucleoside which, for the described system, was on the order of 50 pmol of oligomer.