Guide to Building Custom Synthetic DNA Sequences: Methods, Automation, and Quality Control

How to Build Custom Synthetic DNA Sequences?

Building custom synthetic DNA sequences involves a precise chemical process known as phosphoramidite synthesis, which assembles DNA bases sequentially on a solid support. This method allows for the automated creation of highly specific DNA strands using controlled, cyclical chemical reactions.

Overview of the DNA Synthesis Method

DNA synthesis primarily uses the phosphoramidite method, where chemically modified nucleotides—phosphoramidites—act as building blocks. These nucleotides link together stepwise to form a defined DNA sequence.

The process employs solid-phase synthesis. DNA construction starts attached to an insoluble solid support, enabling efficient washing and reagent exchange between synthesis steps.

Solid Phase Synthesis Setup

Synthesis begins on a small column packed with activated porous glass particles. These particles are coated with linker molecules, which anchor the first nucleotide and enable chain elongation.

Phosphoramidites come as white powders dissolved in acetonitrile, an organic solvent used throughout synthesis both to solubilize reagents and rinse the column.

Stepwise Chemical Cycle for DNA Chain Elongation

The DNA strand grows by repeating a series of chemical steps for each nucleotide: deblocking, coupling, capping, and oxidation. Steps are interspersed with washes to ensure clean reactions.

1. Deblocking: Acid treatment removes the Dimethoxytrityl (DMT) protecting group from the 5′-OH of the growing chain, exposing it for the next nucleotide addition.
2. Coupling: The active phosphoramidite nucleotide and an activator (often a tetrazole derivative) react to attach the new base to the 5′-OH group.
3. Capping: Unreacted 5′-OH groups from failed coupling are acetylated to prevent further elongation, improving final product purity.
4. Oxidation: Iodine oxidation converts the phosphite triester linkage at the coupling site into a natural phosphodiester bond by adding an oxygen atom.

Between these steps, thorough washing with acetonitrile removes excess reagents and avoids side reactions. The chain assembly proceeds from the 3′ end toward the 5′ end of the DNA strand.

Automation in DNA Synthesis

This repetitive process requires hundreds to thousands of precise reagent dispenses per oligonucleotide. Modern synthesizers operate robotically, often handling 96 or 384 samples simultaneously in well plate formats for high throughput.

Post-Synthesis Processing

Once synthesis is complete, the DNA remains attached covalently to the solid support via the base-labile linker. To release the DNA, treatment with a strong base such as ammonia or methylamine cleaves the DNA-linker bond in a process called cleaving.

This base treatment also removes nucleotide protecting groups that were necessary to prevent unwanted side reactions. Removing these groups activates the DNA strands for downstream use, known as deprotecting.

The released DNA solution is collected and the base reagent evaporated. The DNA is resuspended in water and purified using cartridge, high-performance liquid chromatography (HPLC), or polyacrylamide gel electrophoresis (PAGE). Purification removes failure sequences and residual chemical protections, ensuring a clean final product.

Quality Control and Delivery

The purified synthetic DNA is quantified to measure yield. Samples are dispensed into tubes for distribution. Quality control assays verify sequence integrity and purity. Documentation accompanies the delivery to inform end users of sequence quality and concentration.

Summary of the Process

• Phosphoramidite chemistry assembles nucleotides on a solid support stepwise.
• The synthesis cycle includes deblocking, coupling, capping, and oxidation, with washing to prevent side reactions.
• Automation allows parallel, precise synthesis of many sequences.
• Cleaving and deprotecting free the DNA from solid support and remove protective groups.
• Purification methods remove impurities and failure sequences.
• Final product is quality checked, quantified, and shipped with data.

How does the phosphoramidite method build custom DNA sequences?

This method uses chemically modified nucleotides called phosphoramidites. They attach base-by-base to a growing chain on a solid support, allowing precise control over the sequence.

What are the main chemical steps in each nucleotide addition cycle?

1. Deblocking removes protective groups to expose the 5′-OH.
2. Coupling attaches the next nucleotide with an activator.
3. Capping blocks any unreacted ends.
4. Oxidation stabilizes the new bond by adding oxygen.

Why is DNA synthesis done on a solid phase?

A solid support, usually porous glass with a linker, holds the growing DNA. This setup simplifies washing, reaction steps, and allows automation of the synthesis cycle.

How is the DNA strand released from the solid support after synthesis?

Base treatment with ammonia or methylamine cleaves the DNA from the base-labile linker and removes protecting groups, yielding functional DNA strands.

How is purity ensured in synthetic DNA production?

Unreacted ends are capped during synthesis to prevent elongation. Post-synthesis purification using cartridges, HPLC, or PAGE removes failure sequences and residual chemicals for a pure product.