|Oligonucleotide Purification |
All oligonucleotides made at IDT are deprotected, desalted, quantitated (OD260), and lyophilized prior to shipping. Desalting removes cleaved protecting groups, residual synthesis reagents, and very short oligo truncation products. Unless otherwise requested, desalting is done for every oligo made at IDT, even if additional higher-level purification is ordered. Desalting purification is sufficient for routine PCR, sequencing, or probe hybridization applications. Click here for more about Salt-Free Oligos.
However, some amount of truncated oligo will remain in the desalted product unless further purification is undertaken. These truncated species are an undesirable yet unavoidable by-product of any chemical synthesis of DNA. Oligos are synthesized one base at a time extending from the 3' end, which is attached to a solid-support matrix. Truncation occurs when the specified base fails to add to the growing chain at a given cycle. We estimate that each cycle of synthetic chemistry is on average 99% efficient (Eff = 0.990). In other words, about 1% of all extensions fail. Some of these failures are capped and do not participate further in chain elongation. Some continue to elongate. The longest of these truncation products is the (n-1)-mer. The (n-1)-mer is not a single species but rather is a mixture of every possible single-base deletion within the sequence (Chen et al., 1999; Hecker and Rill, 1998; Temsamani et al., 1995). Thus after synthesis and desalting, the final product is a heterogeneous mix of desired full-length oligo and undesired truncated oligos.
Some amount of undesired, truncated product will accumulate with each cycle of chemistry. Therefore, both the synthesis efficiency and the length of the oligo determine the amount of accumulated unwanted oligo in the final product. The fraction of full-length product present after synthesis equals (Eff)n-1 with 'n' representing the number of bases in the oligo. As can be seen in the table below, long oligos are less pure than short oligos. Efficient synthesis is crucial for maximizing yields, particularly with longer oligos.
|0.995 ||0.990 ||0.985 ||0.980 |
|91% ||83% ||75% ||68% |
|78% ||61% ||48% ||37% |
|69% ||48% ||33% ||22% |
|61% ||37% ||22% ||14% |
IDT recommends that all oligos over 40 bases in length receive further purification. For demanding applications - such as site-directed mutagenesis, cloning, and gel-shift protein-binding assays - additional purification is recommended, even for shorter oligos. For example, use of an unpurified oligo in site-directed mutagenesis risks the possibility that a truncated oligo will be cloned instead of the desired full-length product; an undesired deletion may be introduced near the site of the desired mutation. No one would make a solution out of NaCl that is only 70% pure, yet many researchers clone using oligos that are less than 50% pure.
IDT offers preparative-scale purification using denaturing acrylamide/urea gels (PAGE), and HPLC. While both methods increase oligo purity, one approach may be superior, depending on modifications and intended use.
|Length ||Hydrophobicity ||Length |
|20-50% ||40-70% ||30-60% |
|Best enrichment for full-length product ||Best for modified oligos with a hydrophobic group ||Good for purifying large quantities - |
10-µmole or larger-scale orders
|Only for 1 µmole scale or less ||Does not remove (n-1)-mer effectively ||Not good for oligos over 40 bases |
| - All oligos > 50 bases |
| - Unmodified oligos < 50 bases in length used for: |
- All modified oligos with a hydrophobic groups:
- Site-directed mutagenesis
- Gel-shift protein-binding assays
- All phosphorothioate antisense oligos *
- Fluorescent dye labels, such as Cy3, Cy5
- NHS-ester conjugations
| - Large scale synthesis |
- In vivo usage
|* The impurities in phosphorothioate oligos used as antisense agents are toxic in tissue culture or in in vivo applications. IDT requires HPLC purification of all phosphorothioate-modified oligos. |
Oligonucleotide purification should be considered anytime an oligo is ordered for any application other than routine PCR and sequencing. Please call our technical support group at 1-800-328-2661 or e-mail email@example.com to discuss your specific needs.