Though the majority of product will be the desired sequence, standard desalt oligos will contain a heterogeneous population of sequences, and this heterogeneity will increase with increasing length of the oligo. Oligos are synthesized 3'->5', base by base, through a series of chemical reactions. Chemical reactions are rarely if ever 100% efficient; the coupling reaction itself is approximately 99% efficient. So with each base addition, about 1% of the growing oligonucleotide chains will not undergo base extension. After base coupling, we perform a capping step to prevent any truncated molecules from participating in further base addition. Because the capping reaction is slightly less than 100% efficient, a very small percentage of the truncated mutants will remain uncapped and can react during subsequent couplings steps. This leads to the formation of deletion mutants. The resulting oligonucleotide preparation, while comprised mostly of the ordered sequence, is a mixture of the full length oligos, truncated sequences, and sequences with internal deletions.
For exceptionally long oligos, such as our Ultramer®
Oligonucleotides and xGen®
Probes, IDT uses a use a proprietary IDT Ultramer synthesis process, which has an increased coupling efficiency, in fact, the highest coupling efficiency in the industry (99.5%). This high coupling rate allows us to synthesize long oligos (xGen Lockdown Probes are 60–120 bases long) with a high percentage of full-length product. Read more about this topic in the related DECODED newsletter article, Getting Enough Full-Length Oligo?