In the early days of molecular genetics, the idea of custom-designing DNA sequences was almost science fiction. Laboratories in the 1950s and 60s struggled with solution-phase methods that were painstakingly slow and limited in scale. These manual techniques produced only tiny amounts of oligonucleotides, just enough to begin deciphering the genetic code, but far from practical for widespread applications.
The turning point came with Marvin Caruthers’ development of phosphoramidite chemistry, and especially its integration into solid-phase synthesis techniques. This milestone, achieved in the late 1970s and early 1980s, transformed oligonucleotide production from a slow, artisanal process into a rapid, automatable, and highly efficient workhorse for modern science [1].
Today, what once required weeks by hand can now be ordered online and delivered in days − with accuracy, scalability, and versatility that spark innovation across diagnostics, therapeutics, synthetic biology, and beyond.
At its core, oligo synthesis is about building a precise string of nucleotides, one base at a time. Solid-phase synthesis delivers this with speed and control, and here's how:
The process begins with the first nucleotide bound to a controlled pore glass (CPG) or polystyrene bead via a cleavable linker. This stable anchoring ensures each reaction step can be performed and washed without losing material, a key advantage over old-school solution-phase chemistry [2,3].
For each base addition, the following steps are carried out in succession:
Deprotection (Detritylation): A mild acid removes the 5’-protecting dimethoxytrityl (DMT) group, exposing the hydroxyl group necessary for the next coupling reaction.
Coupling: The incoming phosphoramidite nucleotide − activated and ready − is introduced with an activator (commonly tetrazole). This forms a phosphite triester linkage to the growing chain.
Capping: Any unreacted 5’-OH groups are blocked using acetic anhydride to prevent them from participating in future steps − minimizing truncated products.
Oxidation: The newly formed phosphite triester is converted into a stable phosphate linkage using an iodine-based oxidation solution, mirroring the natural backbone of DNA and RNA.
Figure 1 - Phosphoramidite oligonucleotide synthesis cycle
With per-step coupling efficiencies often exceeding 99%, even long oligos can be synthesized with relative ease and high yield.
Once the desired sequence is complete, the oligo is released from the support. Base-protecting groups are removed − typically under basic conditions − yielding a free, functional strand of DNA or RNA in solution.
Crude syntheses may contain truncated or misassembled fragments. This is where quality matters: HPLC or PAGE separates the full-length product, while mass spectrometry, capillary electrophoresis, or UV absorbance (260 nm) verify the oligo’s identity and purity.
Modern DNA/RNA synthesizers streamline this process, allowing hundreds of sequences to be produced daily. Miniaturization, parallelization, and microfluidic advances have slashed reagent consumption and turnaround times, all while maintaining rigorous quality standards.
Invitek Diagnostics knows that high-quality oligonucleotides are more than just reagents − they’re essential tools that accelerate discovery and enable real-world solutions.
Molecular Diagnostics
Short custom primers drive the power of PCR; fluorescent or quencher-labeled probes − such as TaqMan® − add specificity. In rapid isothermal assays, tailored oligos make on-site pathogen detection fast, sensitive, and reliable.
Therapeutics and Gene Regulation
Antisense oligonucleotides, siRNAs, and aptamers are revolutionizing medicine − from correcting splicing errors to silencing disease-causing genes. A growing number of these therapies are now approved for human use, addressing conditions once thought untreatable.
Gene Editing and Synthetic Biology
Synthetic oligos are at the heart of CRISPR-Cas systems − as guide RNAs or donor DNA templates. In synthetic biology, entire genes and genetic circuits are constructed from overlapping oligos, enabling the design of custom pathways and biomolecules.
Research Tools and Genomic Exploration
Microarrays, fluorescent in situ hybridization (FISH) probes, and molecular barcodes all rely on synthetic oligonucleotides. These tools help map gene expression, track genetic variation, and dissect nucleic acid interactions with high precision.
The demands of modern science call for longer, cheaper, and more eco-conscious oligo synthesis. Emerging enzymatic methods leveraging polymerases or terminal transferases offer greener alternatives to traditional chemical approaches, especially for very long sequences.
At Invitek Diagnostics, continuous refinement of synthesis protocols, thoughtful reagent sourcing, and robust quality control systems underscore our commitment to empowering researchers and diagnostic developers. From the lab bench to clinical impact, our oligo synthesis workflows deliver precision, reliability, and innovation.
Find out more about our oligo synthesis capabilities at https://www.invitek.com/en/custom-oligo-synthesis/dna-rna-oligo-synthesis-services
ATDBio. Chapter 5: Solidphase oligonucleotide synthesis. In Nucleic Acids Book – Solidphase oligonucleotide synthesis.
Roy, S.; Caruthers, M. H. Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and HPhosphonate Chemistries. Molecules 18 (11), 14268–14284 (2013). DOI: https://doi.org/10.3390/molecules181114268
Lönnberg, H. Synthesis of oligonucleotides on a soluble support. Beilstein Journal of Organic Chemistry 13, 1368–1387 (2017). DOI: https://doi.org/10.3762/bjoc.13.134