Purification of Oligonucleotides by Ion-pair Chromatography on Hybrid Silica Particles
Synthetic oligonucleotides are used as primers for DNA
sequencing and PCR and are also being investigated as drug candidates.
Due to failure sequences, the purity of 25-mer oligonucleotides is
typically 80-85%. Higher purity is required, especially for PCR
applications. Typically, oligonucleotides are purified by
electrophoresis or HPLC. The current techniques have limitations.
Slab-gel electrophoresis is a laborious process, although it affords
very high purity (>98%). HPLC suffers from the fact that the
oligonucleotides are purified in the trityl on state. After
purification, the DMT protecting group must be removed. To address
these limitations, we have developed methods for trityl off
purification of oligonucleotides using ion-pair reverse phase
chromatography on hybrid silica phases (XTerra MSC18). We demonstrate
purification strategies for synthetic oligonucleotides up to 30-mers on
a micromolar scale.