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Oligonucleotide Purification

The product of oligonucleotide synthesis must be purified to remove unwanted substances and particles that are formed during the synthesis - chemical impurities (protective groups and excessive salts) and contamination with oligonucleotide chains of improper length.

Why are chains of improper length formed during synthesis?

It is characteristic for oligo synthesis that coupling (connecting a new base to the growing oligonucleotide) does not take place with 100% efficiency, not even under optimum reaction conditions. Thus contamination of the product results, with a certain proportion of short incompletely synthesized chains.

In case of long oligos (more than 50 bases), the proportion of full length chains may decline even below 50%. In such a case, purification must be performed, capable of removing shorter incompletely synthesized chains.

Possible ways of oligonucleotide purification

Several methods of oligonucleotide purification exist, differing markedly in their implementation as well as quality of the resulting product.

• Standard purification: Upon deprotection, the oligonucleotide is desalinated in a column usually filled with the medium Sephadex G-25 used for SEC (Size Exclusion Chromatography). The oligonucleotide (as a molecule of high molecular weight) flows through the column; smaller molecules (protective groups, excessive salts etc.) remain in the pores of the medium. Thus most chemical impurities are removed from the product with relatively low losses.
• OPC purification: This type of purification is based on capturing oligonucleotides that at its end 5´ contain the trityl group on the lipophilic sorbent of the OPC column (Oligonucleotide Purification Cartridge). Upon finishing the synthesis of the oligo, the trityl group is present only on completely synthesized chains and double length chains (whose existence has no negative impact during applications), eluated in the pure form (without the trityl group) in the last step of the purification process.
• HPLC purification: This purification method incorporates separation of the oligonucleotide itself upon deprotecting it from all impurities based on the magnitude of its negative charge (molecular weight). This method of purification is very demanding and connected with considerable losses.

Guaranteed and usual yields of individual purification types