Oligonucleotide synthesis

Oligonucleotide synthesis is the non-biological, chemical synthesis of defined short sequences of nucleic acids. It is extremely useful in laboratory procedures covering a wide range of molecular biology applications. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases. Typically, synthesized oligonucleotides are single-stranded DNA molecules around 15-20 bases in length. They are most commonly used as primers for DNA sequencing and amplification, as probes for detecting complementary DNA or RNA via molecular hybridization, and for the targeted introduction of mutations and restriction sites, allowing for the synthesis of artificial genes.

Synthesis substrates

Oligonucleotides are chemically synthesized using phosphoramidites. A phosphoramidite is a normal nucleotide with protection groups added to its reactive amine, hydroxyl and phosphate groups. These protection groups prevent unwanted side reactions and force the formation of the desired product during synthesis. The 5' hydroxyl group is protected by DMT (dimethoxytrityl), the phosphate group by a diisopropylamino (iPr2N) group and a 2-cyanoethyl (OCH2CH2CN) group.

Sequential synthesis

In solid-phase synthesis, the 3' end of the oligonucleotide is bound to a solid support column on which all reactions take place. The 3' group of the first base is immobilized via a linker onto a solid support (polystyrene beads or similar). This allows for easy addition and removal of reactants. In each step, the solutions with the nucleotides for the next reaction are pumped through the column from an attached reagent delivery system and washed out before the next nucleotide is added. In modern synthesizers, reagent delivery and washing steps are controlled via computer based on the desired sequence. At the end of the synthesis program, the oligonucleotide is cleaved off the solid support and eluted from the column.


Microarrays

An interesting development of this technology has allowed gene chips to be made, where the probes are synthesized on the silicon chip, and not printed, allowing a higher resolution. This can be done via a mechanical mask where thin silicon rubber capillaries are put on a glass slide and the probes synthesized. More high-tech versions employ photolayable products and Photolithographic mask or micro mirrors. The 1cm2 surface of silicon is coated with a linker and a photo protecting group such as nitroveratryloxycarbonyl is used and the mask exposes to a lamp the spots that will receive the subsequent nucleotide: this step is repeated for all four bases, but only one correct one is added to the growing probes on each spot