What It’s All About

DNA phosphoramidites are the core building blocks used to make custom DNA sequences—typically 10 to 100 bases long—on a solid support. This approach underlies the production of primers, probes, gene-editing tools, and certain therapeutic oligonucleotides.

These reagents are engineered to couple efficiently and reproducibly. Their standard design includes a 5′-O-DMT protecting group, base-protected nucleosides, and a reactive phosphoramidite moiety, which together enable high coupling yields during synthesis.

This guide focuses on how DNA phosphoramidite synthesis is actually carried out in the lab, based on common practice and widely accepted knowledge. The emphasis is on reliability and repeatability, not detailed reaction theory.

How It Works: The Basics

DNA is assembled step by step on a solid support. Each synthesis cycle adds one nucleotide, extending the chain from the 3′ end toward the 5′ end. Because the growing DNA strand remains attached to microscopic beads, excess reagents can be easily washed away after each step.

Each cycle consists of four core steps:

1.Deprotection
The 5′-DMT group is removed to expose a free hydroxyl group for the next coupling step.

2.Coupling
An activated phosphoramidite reacts with the free 5′-OH, forming a temporary phosphite linkage.

3.Capping
Any unreacted hydroxyl groups are blocked to prevent truncated sequences from continuing to grow.

4.Oxidation (or Sulfurization)
The temporary linkage is converted into a stable backbone. Using sulfurization instead produces phosphorothioate linkages.

The Step-by-Step Guide to DNA Synthesis

What You’ll Need

Solid Support 

• CPG resin (50–100 μmol/g)

Phosphoramidite Monomers

• Standard DMT-protected monomers (e.g., dA(Bz), dC(Bz), dG(iBu), dT)

 Main Reagents

•  3% DCA in DCM (DMT removal)
• Anhydrous acetonitrile (MeCN)
• Tetrazole (activator)
• Acetic anhydride / pyridine (capping)
• Iodine solution (oxidation)
• 28% aqueous ammonia (cleavage and base deprotection)

Equipment

• Manual synthesis column or automated DNA synthesizer
• RP-HPLC with a C18 column
• Lyophilizer

The Process

1.Prepare the Resin
Load ~100 mg of CPG resin and rinse with dry MeCN three times.

2.Remove the DMT Group
Treat with 3% DCA/DCM for about 2 minutes.
Wash thoroughly with MeCN until the orange DMT color disappears.

3.Couple the Nucleotide
Activate the phosphoramidite (≈5 equivalents) with tetrazole (≈10 equivalents) in MeCN.
React with the resin for 10–15 minutes (typical coupling efficiency ≥99%).

4.Capping
Add the capping solution for ~2 minutes to block unreacted sites.

5.Oxidation
Treat with iodine solution for ~2 minutes to stabilize the backbone.

6.Repeat the Cycle
Repeat steps 2–5 until the full DNA sequence is assembled.

7.Cleavage and Deprotection
Incubate the resin with 28% ammonia at 55 °C for ~12 hours.

8.Purification
Filter, concentrate, purify by RP-HPLC (typically >95% purity), and lyophilize to obtain dry DNA.

Tips and Tricks

Phosphorothioate (PS) DNA

Replacing iodine oxidation with a sulfurization reagent (e.g., Beaucage reagent) produces phosphorothioate linkages, which improve resistance to nuclease degradation—useful for therapeutic applications.

Adding Labels

Phosphoramidites carrying fluorescent dyes or functional tags (e.g., FAM, Cy5, biotin) can be introduced at terminal positions to generate probes, diagnostic tools, or affinity reagents.

Handling Reagents

DNA phosphoramidites are highly moisture-sensitive. Store them dry at −20 °C and allow them to warm to room temperature before opening to minimize moisture damage.

For routine or larger-scale synthesis, automated synthesizers provide better consistency. Manual synthesis is generally sufficient for small-scale projects.

FAQ

What’s the best DNA length for this method?

It works most reliably for sequences between 10 and 100 bases. Longer sequences require optimized cycle times and high-purity reagents.

How should synthesized DNA be stored?

Store lyophilized DNA at −20 °C or −80 °C in DNase-free tubes. Avoid repeated freeze–thaw cycles.

Can modified bases be incorporated?

Yes. Modified phosphoramidites (such as 5-methyl-dC) can be incorporated directly during synthesis.

Do I need an automated synthesizer?

No, but automation improves consistency for higher throughput or regulated work.

In Conclusion

DNA phosphoramidite chemistry remains the most reliable method for producing custom DNA with precise sequence control. By keeping reagents dry, following appropriate cycle times, and handling products carefully, high-quality DNA can be produced consistently for research, diagnostic, and therapeutic applications.