# 01 Proteomics experiments
Purification of Oligonucleotides and Fluorescently labeled Oligonucleotides by Ion-pair Chromatography on Hybrid Silica Particles
Reb J. Russell II, Paul Rainville, Eva Budman, and Martin Gilar
Synthetic oligonucleotides are used as primers for DNA sequencing and PCR and are also be 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 upto 1.0 micromolar scale. Additionally, there are fluorescently labeled oligonucleotides that are important in many genomic assays such as detection of Single nucleotide polymorphisms (SNPs). The oligonucleotides used in SNP assays should not only be pure of failure sequences but also of unlabeled full-length and failure sequences.
We demonstrate purification of the synthetic fluorescently labeled full-length oligonucleotides on the micromolar scale.