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Oligonucleotide Synthesis

By Dr Ananya Mandal, MD

Oligonucleotides are short chains of base pairs that are used for a variety of applications in research, genetic testing and forensics.

These oligonucleotides are synthesized to hybridize to specific DNA or RNA sequences in processes such as polymerase chain reaction (PCR), DNA sequencing, library construction and artificial gene synthesis.

Oligonucleotides are built using protected phosphoramidites of natural or chemically modified nucleosides. Non-nucleosidic compounds are also used, but to a lesser extent.

A biochemist at the University of Wisconsin called Professor H. Gobind Khorana was a key figure in the elucidation of the genetic code. Khorana helped establish how phosphate linkages in DNA are formed through enzymatic catalysis in a reversible reaction. Specifically, a deoxynucleotide triphosphate is added to an expanding deoxynucleotide monophosphate polymer through the actions of polymerase in the presence of magnesium. This forward reaction adds one base to the polymer at a time. Khorana used this knowledge to start synthesizing polypeptides in the laboratory.

The assembly of an oligonucleotide chain occurs in the 3’- to 5’-terminus through the “synthetic cycle” process. Each of these cycles results in the addition of one nucleotide residue to the chain. The cycles are repeated until the required number of nucleotides is achieved. However, the process is limited by an incomplete yield of each synthetic step and side reactions that occur. The maximum length of these oligonucleotides therefore does not exceed 200 nucleotide residues.

Reviewed by , BSc

Further Reading

Last Updated: Sep 10, 2014

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