Oligonucleotides Synthesis Cycle - BioMolecules

Oligonucleotides Synthesis Cycle

Synthetic DNA and RNA oligonucleotides are made using phosphoramidite chemistry, a technology which has remained unchanged for more than two decades. The chemical synthesis takes place in the 3' - 5' direction unlike the natural polymerization process which is in the 5' - 3' direction. Assembly of the protected nucleosides to form a oligonucleotide chain is carried out in following four chemical steps: [A]. Deblocking, [B]. Activation-coupling, [C]. Capping and [D]. Oxidation. Each step is followed by a washing step for removing extra chemicals, which are inhibitory of the next step.

After synthesis, all the protecting groups are removed and oligonucleotides in the natural form is obtained. These are then purified in any of the following ways:

Desalting (Reverse Phase Cartridge): This is an efficient process for oligos of length up to 60 mers, which removes all salts, left-over bases, truncated products and toxic leftovers of synthesis. This is a high throughput process and yields >90% purity.

HPLC purification: Depending on the level of purity, three kinds of HPLC purification procedures are available. Reverse phase HPLC gives > 95% purity, Ion Exchange HPLC gives > 95% purity, and orthogonal HPLC (both RP and IE) gives > 98% purity by removing the salts, toxic leftovers and truncated products very efficiently. HPLC Purification is the best method for purification of oligonucleotides of length 30 to 80. However, this is not a high throughput process like desalting.

Gel Purification: This is another purification procedure and is of two types:-

Polyacrylamide Gel Electrophoresis (PAGE) purification, yields oligonucleotides with >97% purity. Salts, toxic leftovers, and truncated products are completely removed using this procedure.
Elchrome Submerged Gel Electrophoresis purification using non-toxic pre-cast gels yields oligos with >98% purity. This procedure completely eliminates truncated products and significantly reduces depurination.

All oligonucleotides synthesized undergo QC check through one of the following methods – Polyacrylamide Gel Electrophoresis, Capillary Gel Electrophoresis, Analytical HPLC or Pyrosequencing, ensuring very high quality molecules.


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