Synthetic DNA oligonucleotides are used broadly across many different application areas, from basic R&D to clinical diagnostics and biotherapeutics. Their meteoric rise to modern-day ubiquity had humble beginnings as a synthesis puzzle for organic chemists, with no clear application in biology . But widespread adoption of recombinant DNA technology in the 1970s opened up and rapidly solidified their place in biology.
Today, DNA oligonucleotides are foundational for genetics and genomics, where they are used for amplification, enrichment, detection, and sequencing techniques . In diagnostics, they are used for the detection of infectious agents and genetic markers of disease. In biotechnology, oligonucleotides can act as therapeutics, guides for genome editing, or building blocks for the assembly of genomes . Finally, biophysics and nanotechnology fields can use oligonucleotides to form intricate 3D structures that self-assemble .
Commercialization of custom oligonucleotide synthesis
With the rapid expansion of oligonucleotides into so many different application areas, there is significant demand for DNA synthesis. Many companies now offer an affordable and fast custom oligonucleotide synthesis service that can accommodate small (i.e., 25 to 50 nmol) or large scales (i.e., 100 mmol). In addition, several different linkages (i.e., phosphorothioate), nucleoside (i.e., 2′-OMe), nucleobase (i.e., inosine), 5′, or 3′ modifications (i.e., dyes, biotin linkers, etc.) can easily be ordered to further functionalize these already versatile biomolecules.
Coupling efficiency and the need for purification
While new oligonucleotide synthesis techniques have been developed in recent years, the methodology that has stood the test of time is phosphoramidite chemistry . Because nucleosides—a deoxyribose (or a ribose in the case of RNA) sugar linked to a purine (adenine or guanine) or pyrimidine (cytosine or thymine) nucleobase—contain several hydroxyl and amino functional groups that can interfere with the stepwise addition of monomers to form full-length DNA oligonucleotides, they can’t be used directly for synthesis. Instead, nucleoside phosphoramidites, which contain specific protective groups to prevent the synthesis of unwanted byproducts, are used as the monomers for oligonucleotide synthesis. Removal of these groups is carefully controlled through an iterative addition of nucleosides, from 3′ to 5′, that involves four chemical steps: deblocking, activation/coupling, oxidation, and capping.
Due to the stepwise deblocking and activation/coupling steps, chemical oligonucleotide synthesis is highly favorable and can be carried out with automated instrumentation, achieving > 99% coupling efficiency for each monomer addition. But no chemical process is 100% efficient and inefficient coupling can cause internal deletions or truncations. As oligonucleotide synthesis progresses and the chain of bases grows, the error probability increases, creating byproducts or failure sequences and decreasing the percentage of desired, full-length oligonucleotide sequence.
The length of the oligonucleotide and/or the use of modifications can increase the likelihood of generating undesirable byproducts. For instance, if a 30mer is required and the average coupling efficiency is 99.9%, the theoretical yield of the desired full-length product will be 97% (0.99930 = 0.97). The theoretical yield continues to decrease as the length of the oligonucleotide increases. For some applications, 3% or greater contamination may be okay, but for applications that require base-level precision such as next-generation sequencing (NGS), removal of truncated or incomplete byproducts through purification of the full-length oligonucleotide can be required.
Choosing an oligonucleotide purification method
For most commercial oligonucleotide suppliers, coupling efficiency is measured in real-time for quality control. Post-synthesis, oligonucleotide quality is typically determined through mass spectrometry or capillary electrophoresis . While these can help identify issues that occurred during synthesis, it does not directly fix these problems.
For that, scientists rely on a variety of post-synthesis purification methods. The oligonucleotide purification technique that you choose will depend on a variety of factors, including the:
- Downstream application
- Desired length
- Presence of specific modifications
- Required yield
Let’s take a closer look at each method and the advantages or drawbacks of each.
Desalting
The process of oligonucleotide synthesis requires the use of a number of chemicals and small molecules that can interfere with many downstream applications. Removing these contaminants is necessary for all applications. It can be done using standard molecular biology techniques, such as precipitation or sizing resins (i.e., G-25 for short oligos; G-50 for longer oligos) . Most commercial vendors offer desalting as a standard part of any oligonucleotide order and desalted oligonucleotides can be used for standard PCR reactions, sequencing primers, hybridization probes, microarrays, or siRNA screening.
Advantages
- Included as standard for most commercial vendors
- Removes small molecule contaminants or inhibitors of downstream applications
- Applicable across all synthesis scales (25 nmol–10 µmol) and oligonucleotide lengths (5–100 bp)
- Necessary for standard molecular biology techniques, including PCR
Drawbacks
- Doesn’t remove failure sequences (i.e., internal deletions or 5′ truncations)
Reversed-phase cartridge purification
Following completion of oligonucleotide synthesis, full-length products can be removed from the solid support. The full-length oligonucleotide can then be separated from failure sequences using a hydrophobic matrix, often available in easy-to-use, column cartridges. The resulting eluted oligonucleotide is deprotected and desalted for further use in any downstream applications.
Many oligonucleotide vendors offer cartridge purification services, but only for certain scales and lengths. Cartridge purification leads to a decrease in yield (often 80% or higher). In addition, as oligonucleotide length increases, there’s an increase in the number of short oligonucleotide products or failure sequences, which often copurify with longer, full-length oligonucleotides, decreasing the overall purity of the eluted oligonucleotide.
Advantages
- Deprotects and desalts full-length oligonucleotides
- Removes failure sequences
- Purity of greater than 80%
- Useful for purification of modified oligonucleotides, with hydrophobic functional groups (i.e., dyes) and applications such as fluorescent sequencing or gel shift assays
Drawbacks
- Doesn’t remove internal oligonucleotide deletions
- Only available for certain scales (50 nmol–1 µmol) and oligonucleotide lengths (7–55 bp)
High-performance liquid chromatography (HPLC) purification
Similar to cartridge purification described above, reversed-phase HPLC purification uses hydrophobicity to isolate full-length oligonucleotides, yet provides higher resolution and purity. It can also be incredibly rapid, even at large-scales, while maintaining exceptional resolution. For that reason and its ability to remove failure sequences and deletions, HPLC is often the gold standard for oligonucleotide purification and is amenable for a large number of precision downstream applications. As with cartridge purification, however, resolution tends to decrease with oligonucleotide length.
Advantages
- Deprotects and desalts full-length oligonucleotides
- Removes failure sequences and deletion byproducts
- Exceptional resolution and purity of greater than 85%
- Useful for purification of modified oligonucleotides, with hydrophobic functional groups (i.e., dyes) and applications such as cDNA library construction, specialty PCR, cloning, mutagenesis, gel shift assays, antisense, and NGS
- Method of choice for large-scale oligonucleotide synthesis required for therapeutic or diagnostic applications
Drawbacks
- Not available for small-scale oligonucleotide synthesis (i.e., 25 nmol)
- More ideal for shorter oligonucleotides (10–55 bp)
- Secondary structures can complicate purification
Denaturing polyacrylamide gel electrophoresis (PAGE) purification
PAGE is the go-to method for establishing base-level resolution across short and/or long oligonucleotides and can routinely achieve greater than 90% purity of the full-length product. What it provides in purity comes at the sacrifice of yield as purification from polyacrylamide is complex and time-consuming. In addition, PAGE purification can be incompatible with select oligonucleotide modifications, including fluorophores, thiols, and a few others [12].
Advantages
- Deprotects and desalts full-length oligonucleotides
- Removes failure sequences and deletion byproducts
- Exceptional resolution and purity of greater than 90%
- Available for a wide range of lengths (7–100 bp) and scales (50 nmol–1 µmol)
- Useful for purification of oligonucleotides being used for cDNA library construction, specialty PCR, cloning, mutagenesis, and NGS
Drawbacks
- Not available for small-scale oligonucleotide synthesis (i.e., 25 nmol)
- Significant reduction in final yield
- Not compatible with certain oligonucleotide modifications
