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Oligonucleotides are a universal ingredient for molecular biology workflows such as PCR and qPCR. For most pipelines, scientists need only small amounts of high-quality oligo to use as primers or barcodes. Unfortunately, the limitations of many conventional synthesis methods preclude oligo manufacturers from producing such small amounts. Instead, they generate significantly more product, requiring researchers to pay for the excess. A new platform from Eurofins Genomics US (Louisville, KY) can produce much smaller volumes, allowing laboratories to order only the amount of oligo they need.
Technology limits
Since the development of DNA synthesis procedures, there have been tremendous improvements, perhaps most notably the ability to produce smaller volumes. Consistent progress has reduced that lower limit to today’s standard volume of 25 nanomoles, but a typical PCR reaction needs just 15 picomoles of DNA. Assuming researchers will run 100 reactions with every set of primers, the current oligo production scale is still an order of magnitude too high. Oligo manufacturers are limited in their ability in this regard. The technical requirements associated with delivering, dispensing, and removing reagents in the synthesis process are obstacles to continued miniaturization. Conventional synthesis technology appears to have reached a plateau for volume reductions.
Balancing act
For end users, the biggest challenge in managing oligo usage is balancing competing needs in quality, quantity, cost, and turnaround time. Some providers offer ultrasmall volumes for specialty oligos or overnight shipping, but these premium services come at a price. Obviously, cutting corners on quality is unacceptable, as that would invalidate any downstream results. Since existing DNA synthesis platforms cannot deliver on all of these factors, however, scientists must select which features they can do without.
DNA used for next-generation sequencing applications must have ultra-low error rates and the highest purity levels. To ensure accuracy and reliability, most oligo manufacturers incorporate meticulous quality-control protocols. Many use mass spectrometry to ensure that the base composition of the DNA sent to a customer matches the order, as well as taking other steps designed to meet rigorous manufacturing standards. Additional reporting measures are required to comply with government regulations for DNA that will be used in clinical labs. It is not surprising that all of these quality-control processes—especially those required for clinical use—increase the cost of oligo production.
To keep costs in check, scientists may try to compromise on other factors, such as turnaround time. This is not always possible, though, and users who need materials quickly may have to pay surcharges for rapid shipping. Vendors may also require customized delivery formats such as oligo pools in specific plates or tubes, or ready-to-use formulations, which can increase costs. The most important factor in managing costs—deciding how much DNA to order—is not feasible in conventional synthesis.
Addressing the oligo challenge
Figure 1 – Eurofins DNA synthesizer.At Eurofins Genomics US, the decision was made to address the oligo volume challenge since this is the most effective way to enable scientists to reduce costs. Company researchers collaborated with a group of aerospace engineers for a fresh perspective on how to achieve this. The pairing made sense, as DNA synthesis incorporates fluidics, air pressure and flow, vibration, and thermodynamics—all important factors in engineering jet engines.
The platform that was a product of this collaboration (Figure 1) delivers high-quality, scalable production volumes and faster manufacturing times. Phosphoramidite chemistry produces coupling efficiencies of better than 99.5%. Miniaturized reagent dispensing enables fast synthesis reactions. Using the new technology, company scientists can produce as little as 1 nanomole of DNA (or as much as needed). This lower oligo volume meets the need for most PCR or qPCR workflows.
Philipp Wenter is vice president of Manufacturing and R&D for Eurofins Genomics US, 12701 Plantside Dr., Louisville, KY 40299, U.S.A.; tel.: 1-800-688-2248; e-mail: [email protected]; www.eurofinsgenomics.com