How scientists build custom DNA strands to power everything from COVID-19 tests to gene therapies
Imagine a machine that can print any sentence you desire. Now, imagine that instead of words, it writes with the fundamental letters of life: A, T, C, and G. This isn't science fiction; it's the reality of oligonucleotide synthesis, a foundational technology that quietly powers everything from modern medicine to genetic engineering.
By building custom strands of DNA and RNA from scratch, scientists can develop diagnostic tests, create new therapies, and probe the deepest mysteries of our genes. This is the story of how we learned to write the language of biology.
Oligonucleotides (or "oligos" for short) are short strands of DNA or RNA, typically ranging from 10 to 200 nucleotides long. Think of them as precise biological phrases or commands.
We synthesize them for countless reasons:
The most famous application. These oligos act as "bookmarks" that define the start and end of a DNA segment to be copied millions of times, a process crucial for COVID-19 testing, genetic fingerprinting, and medical research.
By making overlapping oligos and stitching them together, scientists can build entire synthetic genes from the ground up.
Designed to seek out and bind to specific genetic sequences, these oligos are used to detect viruses, identify genetic mutations, or visualize genes in cells.
"Antisense" oligonucleotides are drugs designed to silence faulty genes responsible for diseases.
The key to all these applications is the ability to create a specific, pre-determined sequence quickly, cheaply, and accurately.
The four nucleotide bases that form the alphabet of life, combined in specific sequences to create oligonucleotides.
So, how do you build a molecule as complex as DNA? The modern method, developed in the 1980s, is a marvel of chemical engineering known as phosphoramidite chemistry. It works on a simple principle: add one DNA "letter" (nucleotide) at a time, in a precise, automated cycle.
The entire process happens on a solid surface—a tiny glass or plastic bead. Here is the step-by-step cycle for building a DNA strand, from first letter to last:
The cycle begins by washing the growing chain with an acid. This removes a protective group (DMT) from the last nucleotide added, "activating" it and allowing it to form a new bond.
A new nucleotide, also protected, is flushed into the chamber. In the presence of a catalyst, it instantly bonds to the activated end of the chain.
A quick chemical wash "caps" the ends of any chains that failed to couple in the previous step. These capped oligos are inert and will be filtered out later.
The new bond formed is relatively weak. An oxidation step converts this bond into the strong, natural phosphate backbone of DNA.
This four-step cycle is repeated for every single letter in the desired sequence. To make a 100-letter oligo, the machine runs this cycle 100 times, with different nucleotides being delivered in the precise order programmed by the scientist.
Typical coupling efficiency is 99.0-99.8% per step, which determines the final yield of full-length product.
Each cycle adds one nucleotide to the growing chain
Let's see how this abstract process translates into a real-world, life-saving application: the development of a PCR test for the COVID-19 virus.
To create a set of DNA primers and probes that can uniquely identify the SARS-CoV-2 virus by targeting a specific region of its N-gene.
The synthesized primers and probes were then used in a real-time PCR (RT-PCR) test with patient samples.
If the SARS-CoV-2 viral RNA was present in a sample, the primers would bind to it, and the enzyme would amplify the target region. During amplification, the probe would also bind and be cut by the enzyme, separating the dye from the quencher and causing a fluorescent signal. The machine detects this fluorescence, confirming a positive test.
The success of this entire diagnostic hinged on the precision and reliability of the synthesized oligonucleotides. An error of even one nucleotide could have led to the test failing to detect the virus or, worse, giving a false positive .
20 nucleotides targeting start of N-gene
20 nucleotides targeting end of N-gene
25 nucleotides with dye and quencher
| Target Oligo Length | Approximate Yield* | Common Application |
|---|---|---|
| 20 nucleotides | ~95% | PCR Primers, Probes |
| 50 nucleotides | ~78% | Gene Assembly |
| 100 nucleotides | ~60% | Complex Probes |
| 150 nucleotides | ~37% | Synthetic Biology |
| Synthesis Scale | Amount of DNA Produced | Primary Use Case |
|---|---|---|
| 50 nmol | ~1-5 ng per base | Research, initial testing of primers |
| 200 nmol | ~10-50 ng per base | Standard primers, probes, small-scale use |
| 1 μmol | ~0.1-0.5 μg per base | Large-scale projects, aptamer screening |
| Error Type | Description | How it's Minimized or Corrected |
|---|---|---|
| Deletion (n-1) | A nucleotide is missing from the sequence. | High-purity reagents, efficient coupling |
| Insertion | An incorrect base is added. | Careful reagent handling, capping step |
| Modified Bases | Bases damaged during synthesis. | Use of stable protecting groups |
Every step of the phosphoramidite cycle relies on a specific set of chemical reagents .
The building blocks (A, C, G, T). Each has a protective group (DMT) to prevent unwanted reactions.
The "de-blocking" reagent that removes the DMT group, activating the chain for the next coupling.
The catalyst that drives the coupling reaction between the activated chain and the incoming nucleotide.
The "capping" reagent that blocks any unreacted chains from growing further, improving purity.
The "oxidation" reagent that stabilizes the new bond, converting it to a natural phosphate backbone.
An ultrapure solvent used to wash the synthesis column between steps and deliver the reagents.
The synthesis of unmodified oligonucleotides has transformed biology from a purely observational science into a creative and engineering discipline. We are no longer limited to just reading the book of life; we can now write new sentences, paragraphs, and even chapters.
From diagnosing a pandemic in record time to designing personalized cancer treatments, the ability to quickly and accurately print custom DNA sequences is a cornerstone of modern biotechnology. It is a powerful tool that puts the very code of evolution itself at our fingertips.