DNA Synthesis in 2025: Technologies, Trends, and Tips

Gene synthesis typically follows a multi-step pipeline:

1. Digital DNA Design: You submit a DNA sequence, often codon-optimized for a target organism.
2. Oligo Synthesis: The sequence is divided into short fragments (∼60–200 bp), which are synthesized chemically or enzymatically.
3. Gene Assembly: These fragments are stitched together into full-length genes using enzymatic or thermal cycling methods.
4. Sequence Validation: Final products are verified by sequencing and delivered in your chosen format (linear fragment or plasmid).

Unlike PCR or cloning, most modern DNA synthesis techniques do not require a physical DNA template—the reaction begins with nucleoside precursors or protected nucleotides and a design file, not existing DNA. In phosphoramidite and array-based methods, synthesis proceeds one base at a time on a solid surface without a DNA template. In enzymatic synthesis, template-free polymerases add nucleotides in a controlled fashion. However, some assembly methods like Polymerase Cycling Assembly (PCA) do require oligonucleotide templates that are assembled via overlapping sequences during PCR.

While this means entirely novel sequences can be built from scratch, it doesn't guarantee all sequences are equally buildable. High GC content, strong secondary structure, and repetitive elements can still hinder success. Additionally, longer sequences are often synthesized in segments and must be assembled either by the provider or by the researcher.

For genes longer than 1 kb, most providers deliver the final product in plasmid format. This makes careful plasmid design critical—both for synthesis compatibility and downstream expression. Modular plasmid architectures that follow synthesis-friendly rules can reduce failure rates, simplify assembly, and support flexible re-use in future builds.

Most gene synthesis still begins with the chemical or enzymatic creation of short DNA fragments known as oligonucleotides, or oligos.

This chemical method sequentially adds protected nucleotide bases to a growing DNA strand bound to a solid support. Each cycle involves coupling, oxidation, capping, and deprotection steps. It's precise and produces high-fidelity oligos up to ~200 bp, but its sequential nature limits throughput. Although solid-phase phosphoramidite chemistry is standard today, oligos were historically synthesized in liquid-phase reactions, which offered higher yields for some applications but were more labor-intensive and less scalable.

Here, thousands of oligos are synthesized in parallel on a silicon or glass chip. Photolithographic or inkjet-based methods remove protective groups with light, allowing selective nucleotide addition. While it excels in scale and cost for libraries, it requires post-synthesis amplification and purification due to higher error rates.

This emerging technique uses engineered DNA polymerases or terminal deoxynucleotidyl transferase (TdT) to add nucleotides one at a time in a controlled, template-free manner. By engineering the enzymes and controlling nucleotide addition with reversible terminators, enzymatic synthesis aims to improve speed, reduce environmental hazards from chemical reagents, and allow longer or more complex sequences to be built.

After oligonucleotides are synthesized, they must be assembled into full-length genes.

Common methods include Gibson Assembly, which uses exonuclease, polymerase, and ligase to join overlapping DNA fragments in a "one-pot" isothermal reaction. It is well suited for modular designs, allowing seamless assembly of multiple fragments for complex constructs. Golden Gate Assembly uses Type IIS restriction enzymes and ligase to join DNA pieces with precise, seamless overhangs, making it ideal for standardized part assembly and combinatorial libraries.

➡️ We previously explored techniques for modular plasmid assembly more closely.

Traditional ligation-based assembly relies on sticky or blunt-end ligation and is generally best for simple constructs where only one or two fragments are being joined.

Polymerase cycling assembly (PCA) is another approach that employs repeated PCR cycles to assemble overlapping oligos into full-length genes. While it is accessible and commonly used in academic settings, it is more error-prone and typically requires downstream correction steps or additional assembly stages for larger constructs.

To improve synthesis fidelity, error correction steps may be applied before or after assembly. These can include enzymatic treatments that recognize and remove mismatches, or deep sequencing followed by selection of correctly synthesized variants. Some workflows integrate high-throughput screening to verify construct integrity.

Assembly and error correction steps have a direct impact on synthesis success. Longer sequences, repetitive motifs, or extreme GC content can lead to increased failure rates or extended turnaround times. Understanding the limits of each synthesis workflow enables more reliable and predictable construct design.

Synthetic biology increasingly requires modified bases or backbones:

• Methylated Cytosines: Common in epigenetics; used to study gene regulation.
• Locked Nucleic Acids (LNAs): Stabilize hybridization; often used in diagnostics.
• Phosphorothioates: Increase nuclease resistance; useful in therapeutic design.
• Non-natural bases: Used in expanding the genetic code or synthetic circuits.

Not all providers support these modifications, and custom synthesis often incurs longer lead times and higher costs. Vendors may require consultation for modified or chemically sensitive designs.

DNA and gene synthesis providers screen submitted sequences to ensure biosafety and compliance:

• Pathogen Screening: Sequences are checked against regulated pathogen databases.
• Dual-Use Oversight: Orders with potential bioweapon relevance may be flagged.
• IGSC Compliance: Most providers adhere to International Gene Synthesis Consortium guidelines.

This screening can delay turnaround or result in rejection of certain sequences. Researchers should anticipate delays when working with pathogen-related genes or sequences derived from regulated organisms.

While many companies offer gene synthesis services, not all use the same technology or excel in the same applications. The table below compares four key players in 2025.

Other emerging companies—like DNA Script, Codex DNA (BioXp), and Evonetix—are focused on benchtop and automated synthesis systems, which may play a bigger role in lab-scale or distributed synthesis in the near future.

Before you submit a DNA sequence for synthesis, make sure it's truly synthesis-ready. Here are a few quick reminders:

• Keep GC content balanced between 30–70%
• Avoid strong secondary structures, direct repeats, and homopolymers
• Codon-optimize for your target organism
• Use modular parts to simplify cloning and future edits

👉 See our in-depth guides on Codon Optimization, DNA Design Tools, and Common Mistakes in Plasmid Design for more tips.

While DNA synthesis has advanced rapidly, several hurdles remain:

• Length and Complexity: Longer sequences or those with repeats/hairpins often fail or require iterative troubleshooting.
• Cost Variability: Projects with challenging sequences can be significantly more expensive due to extra QC steps.
• Error Correction Bottlenecks: Especially for pooled libraries, verification remains a major time sink.
• Turnaround Delays: Even simple orders can be delayed when demand is high or sequences hit design flags.

Several exciting frontiers are reshaping what DNA synthesis can do:

• Benchtop Gene Printers: Devices like BioXp and DNA Script's Syntax enable decentralized synthesis.
• AI Design Tools: Predictive software is being integrated to flag synthesis issues before you order.
• Genome-Scale Synthesis: From synthetic chromosomes to custom cell lines, gene synthesis is scaling up.
• Regulatory Considerations: Dual-use sequence screening and IGSC compliance remain essential for responsible synthesis.

DNA synthesis is more powerful and accessible than ever—but success still depends on good design and the right provider. By understanding how synthesis works and what makes some sequences harder to build, researchers can avoid costly delays and failed constructs.

GenoCAD supports the design of modular, synthesis-ready plasmids. Whether you're building a new expression system or troubleshooting a failed sequence, we can help you get it right the first time.

Need help synthesizing complex sequences? Our team of plasmid experts is ready to assist. Reach out today to streamline your research.