How to Choose the Best Oligonucleotide Purification Method for Your Research

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 [1]. But widespread adoption of recombinant DNA technology in the 1970s opened 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 [2–5]. In diagnostics, they are used for the detection of infectious agents and genetic markers of disease [6]. In biotechnology, oligonucleotides can act as therapeutics, guides for genome editing, or building blocks for the assembly of genomes [7–9]. Finally, biophysics and nanotechnology fields can use oligonucleotides to form intricate 3D structures that self-assemble [10].

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.

Although new techniques have been developed in recent years, phosphoramidite chemistry is the methodology that has stood the test of time for oligonucleotide synthesis [11]. 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 terminal 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.

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 [12]. 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.

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 [12]. 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 [12].

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 preferred method 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.

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].

In addition to the purification methods available for your oligonucleotides, desalting exists as a processing option. The process of oligonucleotide synthesis requires the use of a few 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) [12]. 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.

Given the rapid pace at which basic research, diagnostics, and therapeutics are being done, the need for custom oligonucleotides has never been higher. Whatever the required application, scale, yield, and purity, Thermo Fisher Scientific can provide the oligonucleotide purification method you need. Here are quick accesses to some of our most popular services to support your research:

• Custom DNA Oligos Synthesis Services
• Oligo Large-Scale and Manufacturing Services
• Oligonucleotides, Primers, Probes & Genes

To further investigate the best purification method for you, check out our different configuration methods or send us a message about your custom DNA oligonucleotides.

1. Itakura K, Rossi JJ, Wallace RB. Synthesis and use of synthetic oligonucleotides. Annu Rev Biochem. 1984;53:323-356. doi:10.1146/annurev.bi.53.070184.001543 
2. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487-491. doi:10.1126/science.2448875 
3. Clark MJ, Chen R, Lam HY, et al. Performance comparison of exome DNA sequencing technologies. Nat Biotechnol. 2011;29(10):908-914. doi:10.1038/nbt.1975 
4. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 1993;11(9):1026-1030. doi:10.1038/nbt0993-1026 
5. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016;17(6):333-351. doi:10.1038/nrg.2016.49 
6. Yershov G, Barsky V, Belgovskiy A, et al. DNA analysis and diagnostics on oligonucleotide microchips. Proc Natl Acad Sci U S A. 1996;93(10):4913-4918. doi:10.1073/pnas.93.10.4913 
7. Sun H, Zhu X, Lu PY, Rosato RR, Tan W, Zu Y. Oligonucleotide aptamers: new tools for targeted cancer therapy. Mol Ther Nucleic Acids. 2014;3(8):e182. doi:10.1038/mtna.2014.32 
8. Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-87. doi:10.1126/science.1247005 
9. Church GM, Elowitz MB, Smolke CD, Voigt CA, Weiss R. Realizing the potential of synthetic biology. Nat Rev Mol Cell Biol. 2014;15(4):289-294. doi:10.1038/nrm3767 
10. Zhang F, Nangreave J, Liu Y, Yan H. Structural DNA nanotechnology: state of the art and future perspective. J Am Chem Soc. 2014;136(32):11198-11211. doi:10.1021/ja505101a 
11. Beaucage SL, Caruthers MH. Deoxynucleoside phosphoramidite - A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 1981;22(20):1859-1862. doi: 10.1016/s0040-4039(01)90461-7. 
12. Ellington A, Pollard JD Jr. Introduction to the synthesis and purification of oligonucleotides. Curr Protoc Nucleic Acid Chem. 2001;Appendix 3:. doi:10.1002/0471142700.nca03cs00