Having reliable access to synthetic DNA has never been more important in the biomedical and technological fields. From DNA constructs that enable key enzyme research experiments to oligos used as the foundation of new therapeutic candidates in biopharma to research on gene fragment synthesis, demand for synthetic DNA is soaring.
No wonder, then, that manufacturers of synthetic DNA are working hard to innovate and deliver better products. But isn’t all DNA the same? As it turns out, there are big differences in the type of DNA constructs you can get — informed by factors such as length, complexity, and vector support, among others — that depend heavily on how each manufacturer synthesizes the DNA.
Perhaps the most important difference that has emerged in DNA synthesis recently comes from traditional chemical synthesis methods versus newer enzymatic synthesis methods.
Chemical DNA Synthesis: A Legacy Built Over Decades
Until a decade or so ago, almost all commercial DNA synthesis processes were based on phosphoramidite chemistry. Originally developed in the early 1980s and honed in the ensuing years, chemical synthesis was essential for enabling the use of synthetic DNA in research. This approach produced oligos for cloning, for PCR, and for building genes or plasmids in countless projects that have fueled the life science field as it exists today.
Step-by-Step Chemical Synthesis Workflow
With chemical synthesis, single-stranded DNA is built one base at a time, moving directionally from the 3’ end to the 5’ end. The first base is tethered to a solid substrate, with individual bases added in sequence. Each base is added through a series of steps that involve being deprotected, coupled, capped, and stabilized before the next base is brought in.
Limitations of Chemical Synthesis
Even though chemical synthesis has improved considerably over the years, it appears to be reaching a point of diminishing returns. Due to technical limitations, which are caused largely by an inherently inefficient coupling process, chemical synthesis is very slow and cannot create long or complex DNA. Here’s what that means:
- It can take several minutes to add a single base with the four-step synthesis process.
- Starting oligos might be no longer than 150 bases or so; this is because the reagents used for synthesis actually damage the DNA strand as it grows.
- With such short starting pieces, oligos have to be stitched together to create the longer constructs scientists need. Unfortunately, the assembly process can introduce errors, so the more pieces that are needed for a final product, the greater the chance for a mismatch with the desired sequence.
- Complex DNA covers a number of commonly occurring genomic elements and patterns, such as repetitive DNA, extreme GC content, and strong secondary structures. Having to design sequences around these elements is a hurdle for many researchers.
Another issue with chemical synthesis techniques is that the process requires toxic materials that create a hazardous waste byproduct. With increasing demand for synthetic DNA, the amount of waste produced is a growing concern.
Enzymatic DNA Synthesis: Emerging Methods Challenge Convention
Scientists looking to improve upon chemical synthesis processes have often wondered: why can’t we let biology do what biology does best? The new breed of enzymatic DNA technology aims to harness the power of enzymes that have evolved to build DNA accurately and efficiently. There are many laboratories and companies that have developed enzymatic synthesis approaches, and they use a variety of enzymes and synthesis methods to accomplish their goal.
While enzymatic techniques outperform chemical ones in some areas, there are certain parameters where chemical processes still do better. However, it’s the early days of enzymatic synthesis, and scientists anticipate that there will be substantial opportunities to improve the performance of these new technologies and methods. These are the areas where enzymatic synthesis has pushed well past its chemical predecessor:
- Oligo length: There is a significant range of starting oligo size among enzymatic techniques, but some can produce contiguous oligos of 700 bases or more, significantly reducing the number of pieces needed for long sequence orders.
- Complexity: The use of engineered enzymes allows for greater flexibility in the composition of DNA that can be synthesized. For some enzymatic synthesis methods, the complex elements that challenge chemical methods do not present a manufacturing challenge.
- Accuracy: With high-fidelity enzymatic synthesis and the reduced need for assembly due to longer starting oligos, new techniques can more efficiently create DNA of the desired sequence without introducing errors along the way.
- Speed: While approaches vary, some enzymatic techniques can incorporate a new base in seconds, rather than minutes.
- Hazardous waste: Because enzymatic synthesis builds on naturally occurring methods, it produces no toxic waste.
Areas where chemical synthesis processes still perform better include overall yield and the ability to produce DNA with modified components such as linkers and tags. While costs for enzymatic DNA synthesis were initially higher than those for chemically built DNA products, recent improvements in gene synthesis have allowed some manufacturers to set prices at or even below those charged by vendors using chemical synthesis.
Technical Challenges of Enzymatic Synthesis
Despite remarkable progress, enzymatic DNA synthesis still faces hurdles. For example, enzymes like TdT (terminal deoxynucleotidyl transferase) can be prone to unintended additions, creating impurities. Secondary structures in DNA can also interfere with enzymatic activity, especially when working with longer sequences or complex motifs.
To address this, researchers are developing modified nucleotides and engineered enzymes that can provide higher fidelity and control.
The Upshot: Chemical vs. Enzymatic Synthesis
With faster processes, longer starting oligos, and greater efficiency and accuracy, enzymatic DNA synthesis techniques are able to support innovation in the life sciences better than chemical synthesis ever could. Enzymatic techniques are a natural fit for scientific projects where time is of the essence — such as designing new mRNA vaccines to respond to emerging health threats, or enabling faster testing and iteration of synthetic biology programs. While benefits will certainly be seen for scientists in academia — especially those using CRISPR and other gene editing workflows — the biggest impact is expected to be felt in pharmaceutical and biotech companies, where more reliable custom DNA synthesis products can accelerate drug discovery and development for some of the most promising classes of therapeutics, like cell and gene therapies.
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The Future of DNA Synthesis: Enzymatic Innovation with Ansa
While chemical synthesis laid the groundwork, enzymatic DNA synthesis technology is redefining what’s possible in speed, accuracy, and reliability. At Ansa, we’re translating these breakthroughs into dependable gene synthesis services that help scientists, biotech, and pharma teams accelerate discovery and therapeutic development. By combining cutting-edge enzymatic technology with our commitment to reliability, Ansa is setting a new standard for the future of DNA synthesis.