From synthesis to sequencing: how synthetic DNA is shaping genomics

What role does synthetic DNA play in modern genomic research? How could you go beyond long-reads to reads of unrestricted length to revolutionise your quality control (QC)? As researchers push the limits of genome analysis, synthetic DNA offers a powerful tool for synthesising mRNA, which is becoming a valuable resource in genomic research and holds huge potential for medical treatments, including vaccines and cancer immunotherapies.

In this interview, Galit Meshulam-Simon, Elegen (United States), explores the potential of ENFINIA Cell-Free Synthetic DNA to unlock new experimental possibilities, as well as how Elegen uses nanopore reads to enable fast turnaround times and confident quality assurance.

What is synthetic DNA, and what is it used for?

In processes like DNA and RNA synthesis, cloning, or amplification, synthetic DNA is a template that guides the accurate construction or copying of specific genetic sequences¹. They provide accuracy and precision, scalability, and fast research and development in fields such as gene synthesis, therapeutic development, and vaccine design.

For example, in vitro transcription (IVT) templates are pre-designed double-stranded DNA sequences that serve as a blueprint or starting point for making new messenger RNA (mRNA) molecules². Alongside the target gene, IVT templates include regulatory elements and a 3’ poly(A) tail to ensure stability and translation efficiency of the resulting mRNA and protein inside cells.

Elegen uses a patented cell-free cloning technology to synthesise longer, faster, and more complex double-stranded DNA than conventional gene synthesis providers offer. We produce full-length linear DNA templates that are free from endotoxins and bacterial DNA contamination, and can support complex sequences, including GC-rich regions, repeats, and structured elements.

In recent years, mRNA therapeutics have gained momentum. What makes them so powerful?

Since the 1990s, mRNA has been studied as a therapeutic tool¹. These studies enabled the rapid development of the COVID-19 mRNA vaccines, and their adaptation to emerging SARS-CoV-2 variants^3,4 due to the ability to rapidly test and redesign sequences.

As mRNA can encode virtually any protein without being integrated into the genome, it is incredibly versatile for vaccines, cancer immunotherapies, and protein replacement therapies. Its use is expanding into many therapeutic areas, including treatments for cancer, autoimmune disease, and rare genetic disorders⁵.

What’s really driving adoption of mRNA as a therapeutic modality is speed: teams can rapidly design, develop, and scale advanced therapies. At Elegen, we support accelerated pipelines with ENFINIA IVT Ready DNA: reaction-ready, next-generation sequencing (NGS)-verified DNA templates for IVT-mediated mRNA synthesis.

Plasmids have traditionally been used for IVT templates. Where do they fall short?

Using plasmids as DNA templates for IVT is one of the biggest bottlenecks in mRNA development because it relies on cloning in bacteria, which introduces long timelines. Additionally, this method harbours the risk of poly(A) recombination during bacterial growth, resulting in a shortening of the average poly(A) length and an increase in intermolecular heterogeneity. Plasmid propagation also involves growth of large cultures, bacterial harvesting, lysis, and multiple filtration steps that are required to prevent contaminants, such as endotoxins or carryover of bacterial genomic DNA⁶.

The inherent complexity of these processes, combined with the additional purification steps required after plasmid linearisation, often results in significant batch effects, adding unnecessary time and variability.

Poly(A) tails are critical for mRNA function. What’s the advantage of encoding them in DNA?

The 3’ poly(A) tail is vital for mRNA stability and efficient translation in vivo⁷. The tail protects the mRNA from exonucleolytic degradation, enhancing transcript stability and thereby supporting higher protein expression⁸. In nature, the number of A residues is variable, typically ranging from 30–160 nucleotides (nt) in mammals⁶. For synthetic mRNA, longer poly(A) tails generally enhance mRNA stability and translation efficiency.

Poly(A) tails can be introduced into IVT RNA either co-transcriptionally by encoding the tail in the DNA template, or post-transcriptionally by treatment with E. coli Poly(A) Polymerase. Enzymatically added tails exhibit broader length distributions than plasmid-encoded tails, but the latter are unstable to maintain, especially with long poly(A) sequences of more than 80 bp⁹.

Encoding the poly(A) tail directly into the DNA template, such as with ENFINIA IVT Ready DNA, enables precise tuning of tail length, typically in the 70–130 nt range, to match the needs of different applications or cell systems.

A tighter distribution of tail lengths leads to more uniform mRNA behaviour, improving ribosome recruitment and translation efficiency across the population¹⁰. Additionally, removing the enzymatic tailing step simplifies the workflow, reduces variability, and improves reproducibility, which is especially important for automation and tech transfer.

What led Elegen to explore Oxford Nanopore sequencing?

Before the launch of our ENFINIA Linear DNA product, the market was largely optimised around short fragments, moderate complexity, and inferred quality based on partial sequencing. We saw an opportunity to deliver longer constructs and higher sequence complexity with near-clonal-level accuracy.

That positioning created a new technical requirement for us. If we were going to claim that level of quality, especially across long, complex sequences, we couldn’t rely on traditional short-read sequencing approaches that validate DNA in pieces and reconstruct the whole assembly computationally. We needed a way to directly verify full-length DNA molecules end-to-end, ensuring that every base, structural element, and repeat region was exactly as designed. This becomes especially critical as you move into constructs with high GC content, repeats, secondary structures, or therapeutic elements, where even a single error can impact downstream performance.

That’s where technologies like Oxford Nanopore Technologies became our focus. Nanopore sequencing allows us to read entire DNA molecules in a single pass, rather than stitching together fragments. This capability enables what we call true sequence verification at the molecular level.

How did Oxford Nanopore sequencing perform in your evaluation of long-read sequencing technologies?

We took a rigorous, data-driven approach to validating nanopore sequencing within our workflow. Internally, we benchmarked against three critical metrics: per-base accuracy, full-length sequence concordance, and detection of low-frequency variants. These are the same quality thresholds that underpin our ~99.999% accuracy standard and our ability to deliver near-clonal DNA without relying on cells.

We also validated these results externally through our beta partners, who were working with a diverse set of sequences, including long and complex constructs. The consistency between our internal data and the performance observed by beta users gave us strong confidence that what we were seeing in the lab would translate into real-world applications.

We also cross-validated using orthogonal methods, including short-read sequencing and functional downstream assays, to ensure that the sequence accuracy we were measuring aligned with actual construct performance. What stood out with the technology from Oxford Nanopore was its ability to provide true end-to-end validation of full-length molecules, enabling us to detect not only base-level errors but also structural variation and low-frequency variants that other methods can miss or obscure.

Ultimately, Oxford Nanopore Technologies is now a key part of our sequencing stack, combining high quality with speed to enable confident QC and fast turnaround time. As part of our multi-platform NGS approach for verifying ENFINIA DNA, we integrate nanopore sequencing and complementary technologies to validate full-length molecules and ensure base-level accuracy. This strategy enables end-to-end verification of complex constructs with high fidelity, delivering DNA with the accuracy, consistency, scalability, and speed required for modern applications.

Why are more teams choosing to outsource DNA production?

It ultimately comes down to speed, quality, and focus.

Building and maintaining an in-house DNA cloning capability requires meaningful capital investment and ongoing operational overhead. You need lab infrastructure, automation (liquid handlers, thermocyclers, colony pickers), reagents, QC workflows, and skilled scientists to design, troubleshoot, and execute cloning strategies. Even for well-resourced organisations, this becomes a continuous investment in both equipment and headcount, with no guarantee of throughput or success rate, particularly as sequence complexity increases.

For smaller biotech and emerging biopharma companies, that investment is often prohibitive. Even in larger organisations with established cloning workflows, complexity becomes a limiting factor. Sequences with repeats, high GC content, secondary structure, or viral elements can break standard cloning systems. At the cutting edge, this quickly slows down development programs and ties up valuable internal resources.

With Elegen, customers get NGS-verified DNA as little as a week, with the ability to reliably produce long, complex constructs via a fully automated, cell-free manufacturing platform without having to build or maintain it themselves. The result is that internal teams can stay focused on what drives value rather than troubleshooting cloning workflows.

This shifts how teams operate. Instead of validating or troubleshooting constructs, scientists can move forward with confidence that the DNA is exactly as designed, reducing rework and downstream failure.

Are you ready to explore how reads of unrestricted length could streamline your QC? Download our brochure to start today.

Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.