Why purify your oligonucleotides?
Oligonucleotides are commercially synthesized by adding each base, one at a time, using chemical reactions. However, this process is not 100% efficient. The resulting product contains a mixture of full-length and truncated oligonucleotides, as well as a small amount of impurities left over from the synthesis process. Therefore, newly synthesized oligonucleotides undergo a purification step to eliminate the impurities and the less-than-full-length sequences. The type of purification you choose should be determined by your application, oligo length, potential modification(s), and the desired yield.
What is cartridge purification?
A common purification method is cartridge purification. It relies on capture of oligonucleotides that have a hydrophobic trityl group on their 5′-end. Normally the trityl protecting group is removed as the last step in each cycle to add a nucleotide to the oligo, but for cartridge purification, the trityl group is left on the last nucleotide as a “chromatographic handle”. As a result, material retained on the column is enriched for the full-length product; truncated species missing the trityl group are flushed through. The bound oligonucleotides are then eluted in an organic solvent, and the trityl group is removed by acid treatment.
Can acid treatment damage my oligos?
Acid treatment performed on the oligo synthesizer is rapid and controlled. However, with cartridge purification, the acid treatment is less controlled and often damages some of the oligonucleotides. Acid treatment can introduce strand cleavage via a process known as acid depurination. In fact, these deleterious effects can be so severe that the quality of these cartridge-purified oligonucleotides can be worse than the unpurified oligonucleotides. We call this “cartridge depurification”. Depurination is detectable only when QC is performed; however, many manufacturers offering cartridge purification do not perform QC.
How does standard desalting differ from cartridge purification?
IDT does not use cartridge purification. Instead, we use a proprietary desalting technique to remove some of the truncation products and small organic contaminants from the synthesized oligonucleotide preparation. As shown in Figure 1A, our standard desalted oligonucleotides yield an equal or higher proportion of full-length oligos compared to cartridge-purified oligos supplied by 3 different manufacturers. In addition, our desalted oligos contain the lowest amount of n–1mer truncated fragments compared to cartridge-purified oligos from these same 3 suppliers (Figure 1B).
Figure 1. IDT proprietary, standard desalting process consistently delivers a high proportion full-length products. The quality of IDT standard desalted oligos was compared with that of cartridge-purified oligos supplied by 3 different manufacturers. Randomly selected oligonucleotides (n = 8) from each manufacturer were analyzed for full-length product and for n–1mers. (A) Purity of cartridge-purified oligos ranged from significantly worse to marginally better than IDT standard desalted oligos. (B) However, cartridge purification method increased the proportion of undesirable n-1mer oligo species in oligos from all 3 manufacturers.