Each cycle of chemical synthesis proceeds with a finite coupling efficiency. The first base is attached to the solid support which means a 20mer requires 19 coupling reactions and a 200mer requires 199 coupling reactions. Overall synthesis yield of full-length product is a function of both coupling efficiency and length according to the relationship:
Full-length product = (eff)(n–1)
where eff = coupling efficiency (for example, 99.5% = 0.995) and (n–1) is the number of coupling reactions needed to make an oligo of length n. The relationship between the percentage of full length product and the coupling efficiency is shown in Figure 1.
As demonstrated in Figure 1, the only way to synthesize a 200mer oligonucleotide with an appreciable yield of full length material (>30%) is to have a step-wise coupling efficiency >99.25%.
Typically, mass spectrometry is considered the gold standard for oligonucleotide synthesis QC. MALDI mass spectrometry is effective for products in the 10–50 base range. Above 50 bases, ESI (electrospray ionization) mass spectrometry can be employed and has an accuracy of better than 0.02% relative mass. Even with the sensitivity and accuracy of ESI-MS, performing QC on long oligonucleotides is complex. If a synthesis preparation is too impure, the various failure products will obscure the mass spectra, making it difficult to deconvolute and identify the desired species. Using our proprietary mass spectrometry methods, we can confidently synthesize and provide ESI-MS quality documentation for oligonucleotides up to 200 bases in length. ESI mass spectra for two Ultramer oligos are shown in Figure 2.