Oligonucleotide synthesis is a very efficient and high-yielding process. Typical yields of oligonucleotide reactions carried out on solid support range from 98 to 99.5% per coupling step. In a typical multi-step oligonucleotide synthesis, impurities accumulate and the overall yield of even a modest sized 21-mer oligonucleotide can range from 67 to 90%, with longer chain oligonucleotides giving correspondingly lower yields.
For researchers, it is often necessary to work with materials of higher purity than are available from crude synthetic mixtures. For this reason, oligonucleotides used for gene knockout, genotyping, and diagnostic purposes are typically purified following synthesis. Few economically viable solutions exist for lab-scale purification of oligonucleotides, and those that do exist – such as ion-exchange chromatography and polyacrylamide gel electrophoresis – are often cumbersome and/or time-consuming.
In this application note, we describe a cost-effective and rapid method for the purification of modest quantities of material, up to 140 nmoles in a single injection, with final purities of greater than 95% using the Waters® ACQUITY UPLC® System with Oligonucleotide Separation Technology (OST) Column chemistry. The purification scale presented matches well with typical oligonucleotide synthetic scales (50 to 250 nmol). The method described below allows for the purification of oligonucleotides with high purity products in 15 to 30 minutes.