Get Help Sign In
ProcessingProcessing

Getting enough full-length oligo?

Coupling efficiency matters!

The coupling efficiency achieved by an oligonucleotide manufacturer has a direct effect on the quality of the oligonucleotides produced. Find out why coupling efficiency should be important to you.

The coupling efficiency achieved by an oligonucleotide manufacturer has a direct effect on the quality of the oligonucleotides produced. When the coupling efficiency is high, there are fewer truncated products and deletions, resulting in a higher percentage of the desired full-length oligo sequence. IDT employs proprietary synthesis techniques and vertical integration, which results in very high coupling efficiencies. The coupling efficiency for standard oligos from IDT is 99.4%. The high coupling efficiency allows for synthesis of a 40-base standard oligo that has approximately 80% full-length product; the same oligo synthesized at an average coupling efficiency of 98.5% might only provide the customer with 55% full-length product at most (Figure 1). Thus, what may appear as small difference in percentage translates to a dramatic difference in the amount of full-length product delivered to the customer. 

High coupling efficiency for long oligos

The theoretical relationship between full-length product yield, DNA oligo length, and coupling efficiencies. Small increases in coupling efficiency (≤1%) result in measurable increases in full-length product yield. The curves represent the theoretical yield for different lengths of oligos based on a coupling efficiency of 99.4% (IDT oligos) and 98.5% (other supplier) using the formula

Full length product = (eff )(n-1)

where eff = coupling efficiency (for example, 99.4% = 0.994) and (n–1) is the number of coupling reactions needed to make an oligo of length n

IDT offers Ultramer™ DNA Oligonucleotides that are synthesized with enriched chemical reactions to have an even higher coupling efficiency of 99.6%. When oligos longer than 100 bases are required for applications such as site-directed mutagenesis, in vitro transcription, or CRISPR genome editing, the small increase of 0.2% in coupling efficiency in comparison to our standard oligos means the percentage of full-length product is increased. In fact, IDT offers oPools™ Oligo Pools which are pools of custom single-stranded DNA sequences in one tube with lengths of up to 350 bases. In comparison, a 98.5% coupling efficiency would yield virtually no full-length product lengths beyond 250 bases (Figure 2).
Highest coupling efficiency, longest oligos

The theoretical relationship between full-length product yield, Ultramer DNA Oligo length, and coupling efficiencies. Ultramer DNA Oligos and oPools Oligos Pools are synthesized with an enriched cycle and proprietary solid supports. The coupling efficiency of 99.6% ensures that these products contain a higher percentage of full-length product than would be offered by another supplier with a coupling efficiency of 98.5%.The curves represent the theoretical amount of full-length product based on the formula in the previous figure.

 

A higher coupling efficiency means better performance

Why should coupling efficiency be important to you? Higher coupling efficiency results in more full-length oligo, and this translates to more consistent experimental results with less batch-to-batch variability. Thus, when placing an order to replace an oligo, you can be confident that the new oligonucleotide will function the same as an oligo from a previous synthesis. Consistency in products means consistency in your reactions and decreasing efforts needed to troubleshoot anomalous results.

IDT proprietary synthesis platforms yield the highest quality modified and unmodified oligonucleotides across various lengths. Even when these oligos are ordered without additional purification, the large percentage of full-length product can provide a substantial improvement in the data you collect.

The presence of truncated oligonucleotides in your product can influence many common molecular techniques, including cloning, mutagenesis, sequencing, and quantitative PCR (qPCR). IDT recognizes that even modest gains in coupling efficiency lead to higher percentages of full-length oligonucleotides in our products and, therefore, improved sensitivity and consistency in your experiments.  

Published Sep 29, 2017
Revised/updated Nov 12, 2020