Oligo Synthesis and Its YieldIn vitro synthesis of oligonucleotides at GENERI BIOTECH is performed using the so called phosphoramidite method whose principle consists in cyclic blocking of reactive groups of a growing oligonucleotide by protective molecules and their subsequent uncovering in order to connect other bases. The resulting product has no phosphate group at none of both ends. The process runs in the opposite direction compared to in vivo methods (thus from the 3´ end to 5´ end), on a solid base that carries the anchored 3´ end nucleotide. Oligos are synthesized from nucleotide precursors carrying the trityl group at the 5´ end. Detritylation of the growing oligonucleotide chain, connection of a new nucleotide (coupling), oxidation of the created phosphite bond to the phosphate one, and blocking of 5´ end groups of the growing oligo that did not react (capping) takes place in each cycle of the synthesis that is composed of about 100 steps. The process can be completely automatic and is executed using the so called synthesizers.
Yield of the SynthesisOligo synthesis is characterized by the fact that coupling (connecting a new base to the growing oligonucleotide) does not provide 100% efficiency, not even under optimum reaction conditions. Product contamination with a certain proportion of short chains with unfinished synthetization is the result. At GENERI BIOTECH, 99% efficiency of the synthesis is normally achieved. Table: Oligo synthesis yield upon 99% coupling efficiency
| | Oligo length | Product content | Impurities content | | 10 bases | 91 % | 9 % | | 20 bases | 83 % | 17 % | | 50 bases | 61 % | 39 % | | 75 bases | 48 % | 52 % | | 100 bases | 37 % | 63 % |
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Relatively lower content of oligonucleotides that show proper length should be expected when ordering synthesis of long oligos (more than 40 bases). GENERI BIOTECH provides OPC or HPLC oligo purification for such cases. The synthesis yield depends not only on the chain length, scale of the synthesis and the applied purification method, but also on the chain composition. Chains with high G and A content, which show lower coupling, provide a markedly lower yield than chains with high C and T content. The lowest yield is provided by pure polypurine chains.
Modified Oligos Synthesis Modified molecules are usually connected to the oligonucleotide chain by means of a spacer that contains several carbons in order to prevent undesirable steric interference.
The 3´ modifying group is inserted on the oligo using the synthetization column that needs to be manually produced in the smallest 200 nmol scale as a rule. The 5´ modifying group is inserted on the oligo after the chain synthesis as the last step of the synthesis.
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