by Dan Gibson, PhD, CTO at Codex DNA
Scientists will soon have the opportunity to participate in the decentralization of one of the most important processes for life science research and the discovery and development of therapies, diagnostics, and vaccines — gene synthesis.
While the capability of synthesizing DNA and RNA is universally recognized as essential in the research community, there has been considerably less consensus about who should own it. Decades ago, DNA synthesis was performed by scientists in their own labs. The process was painstaking, error-prone, and risky due to the need for toxic chemicals. When service providers emerged and offered to relieve labs of this onerous task, scientists were glad to embrace outsourcing.
For years, outsourcing has been standard practice for nearly all DNA synthesis needs. But this arrangement came at a price: long turnaround times that may extend to weeks or months, as well as high costs for higher-quality products. Despite the recent era of incredible innovation in genomic technologies, customers of these service providers have seen lag times and costs remain stubbornly fixed.
Now, though, gene synthesis is coming full circle with a fleet of new technologies designed to restore this capability to individual laboratories. After decades of commitment to centralization, these enzymatic DNA synthesis tools offer a means of democratizing access to DNA and RNA synthesis. They represent a novel approach that eliminates the previous pitfalls of hazardous chemicals and labor-intensive work, replacing them with automated instruments that produce DNA or RNA on-demand.
Current enzymatic DNA synthesis technologies will need considerable improvement to achieve acceptable cost and accuracy levels, but a hybrid approach may offer a faster path to bringing DNA synthesis back to the bench, where it can accelerate research experiments or pharmaceutical development and give scientists greater control over their work.
Challenges with Conventional Synthesis
For scientists relying on traditional approaches to gene synthesis — either through service providers or through a couple of old-school bench techniques that have remained in use — there are three common challenges.
The first is accuracy. Even the best of today’s widely used synthesis technologies do not achieve perfect coupling efficiencies. The cyclical process of building synthetic DNA means that even small inefficiencies are compounded over time into higher error rates. For the construction of a 100-base product, for example, a less-than-perfect coupling efficiency could mean that overall accuracy for the resulting oligo is only about 60 percent. That low fidelity is simply not tolerable in scientific experiments or workflows where even a single wrong base can cause failure.
Overcoming the accuracy problem leads to the second major challenge: cost. Scientists typically deal with fidelity issues by cloning into E. coli and sequencing the assembled genes derived by those oligos. Both approaches add significantly to the cost and time needed for the synthesis part of the workflow. Even if those steps can be avoided, chemical DNA synthesis is a costly endeavor. At a standard cost of 10 cents per base, getting enough oligos to build a gene would cost somewhere in the neighborhood of $300. Synthesizing a small genome could take $200,000 or more. With most scientists needing to test many different genes derived from these oligos — unsure whether a specific DNA sequence will achieve a desired goal — these synthesis costs are out of reach for most labs.
The final challenge stems from the hazardous waste produced by most synthesis processes. To build one or two genes, most scientists would need to order a 96-well plate of oligos. That single purchase would generate several liters of hazardous waste. Today, scientists are faced with two bad choices — either using a service provider, which is capable of properly disposing of the waste but still generates it, or handling synthesis in-house and having to deal with all those liters of hazardous waste themselves.
Enzymatic DNA Synthesis
Recently, new technologies have emerged in this space based on enzymatic DNA synthesis rather than chemical synthesis. The use of biological rather than chemical reactions avoids the toxic materials that lead to hazardous waste. Because of that, these enzymatic approaches are finally allowing scientists to reconsider outsourcing their DNA synthesis capabilities. The opportunity to construct needed oligos on-demand, in-house would be important for accelerating research and drug discovery efforts.
Traditional DNA synthesis is performed with phosphoramidite chemistry in reactions where bases are added sequentially to create the desired product; this technique produces oligos of fairly high quality and generally low cost. By contrast, enzymatic DNA synthesis involves the use of natural or engineered enzymes to connect nucleic acids together in the appropriate sequence.
Enzymatic DNA synthesis techniques are still young enough to require further optimization before they’re ready for widespread use. Most are based on the naturally occurring enzyme terminal deoxynucleotidyl transferase (TdT), which is used in biological systems to stitch new bases onto a sequence of DNA. While TdT-based methods are a huge improvement in avoiding the production of hazardous waste, they currently suffer from high costs and low fidelity.
The TdT enzyme is not terribly efficient. At the moment, that means scientists must add more reagents to build their target sequence, increasing costs for the entire synthesis process. TdT-based synthesis products are an order of magnitude more expensive, per base, than traditional synthesis products. In addition, the TdT enzyme is overly permissive, so it adds the incorrect base more often than it should. As a result, TdT-based oligos have more sequence errors and may require the same or even more post-synthesis quality control steps that add to the time and cost of traditional synthesis.
Despite these drawbacks, TdT-based versions of enzymatic DNA synthesis have tremendous potential for bringing DNA synthesis back to the bench. Many other genomic technologies have started with inefficient, lower-performing enzymes and improved significantly with years of development and enzyme engineering. There is every reason to believe that fine-tuning the TdT enzyme or replacing it with a higher-performance enzyme will in time make this kind of DNA synthesis a viable alternative to conventional techniques.
However, a hybrid approach may offer the most immediate benefit for scientists interested in performing their own gene synthesis. By combining traditional synthesis with an alternative approach to enzymatic DNA synthesis, scientists can build high-quality, low-cost oligos without generating any hazardous waste in the lab.
Here’s how it works. The process begins with the one-time construction of a universal library of short, five-base oligos using traditional phosphoramidite chemistry at a central manufacturing facility. These 5-mers serve as building blocks for the next phase, which is based on an enzymatic synthesis technique performed by an automated benchtop instrument. Unlike TdT approaches that add a single base at a time, this method ligates short oligos together using enzymes that have been used in laboratories for decades. Oligos can be stitched together over and over to build genes or even genomes.
This hybrid version of enzymatic DNA synthesis offers several advantages. Because it starts with 5-mer building blocks, it takes fewer reactions to build longer oligos; this significantly reduces the risk of sequence errors and results in highly accurate constructs. Short-oligo ligation is also more cost-effective. The starting library is built with traditional, low-cost chemical synthesis, so upfront costs are kept low. In the enzymatic part of the synthesis workflow, unused 5-mers remain in the library, allowing the synthesis instrument to access them again and again over the course of millions of cycles of DNA synthesis. Hazardous waste created by a synthesis vendor is minimized — needed only during production of the initial library — and restricted to a facility with proper disposal techniques. In the lab, scientists use only enzymatic DNA synthesis on an automated instrument, which leads to harmless aqueous waste.
Looking Ahead
For too long, scientists have been forced to delay experiments due to the long turnaround times for getting synthetic DNA and the multitude of quality-control steps required to ensure the resulting constructs contain the correct sequence. This centralized synthesis model is slowing the pace of scientific discovery and development.
Enzymatic DNA synthesis approaches will finally make it possible to put this capability back in the lab where it belongs, giving researchers control of their DNA products with much faster results. While TdT-based synthesis approaches may require several more years to be commercially attractive, a hybrid method for short oligo ligation and assembly could realize the promise of enzymatic DNA synthesis in the near future.
About the Author: Dan Gibson, creator of the industry-standard Gibson Assembly® method, is Chief Technology Officer at Codex DNA.