Long Oligodeoxynucleotides: Chemical Synthesis, Isolation via Catching by Polymerization, Verification via Sequencing, and Gene Synthesis and Expression Demonstration

09 October 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Long oligodeoxynucleotides (ODNs) are segments of DNAs having over one hundred nucleotides (nt). They are typically assembled using enzymatic methods such as PCR and ligation from shorter 20 to 60 nt ODNs produced by automated de novo chemical synthesis. While these methods have made many projects in areas such as synthetic biology and protein engineering possible, they have various drawbacks. For example, they cannot produce genes and genomes with long repeats and have difficulty to produce sequences containing stable secondary structures. Here we report direct de novo chemical synthesis of 400 nt ODNs, and their isolation from complex reaction mixture using the catching-by-polymerization (CBP) method. To determine the authenticity of the ODNs, 399 and 401 nt ODNs were synthesized and purified with CBP. The two were joined together using Gibson assembly to give the 800 nt green fluorescent protein (GFP) gene construct. The sequence of the construct was verified via Sanger sequencing. To demonstrate the potential use of the long ODN synthesis method, the GFP gene was expressed in E. coli. The long ODN synthesis and isolation method presented here provides a pathway to the production of genes and genomes containing long repeats or stable secondary structures that cannot be produced or are highly challenging to produce using existing technologies.


gene assembly
long oligonucleotide
automated synthesis
synthetic biology
oligonucleotide purification
tagging phosphoramidite

Supplementary materials

Long oligo synthesis
Experimental details, ODN sequences, PCR primers, and sequence alignments


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Comment number 2, Shiyue Fang: Nov 15, 2023, 21:03

Website for the Polymerizable Tagging Phosphoramidites such as Compound 2 in the Preprint is at https://chem.sites.mtu.edu/amidities/. All other materials needed for long oligo synthesis and purification as well as for gene synthesis are available from other companies.

Comment number 1, Shiyue Fang: Oct 13, 2023, 14:59

for information about the availability of the polymerizable tagging phosphoramidite, contact AMIDITES at [email protected]