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The efficiency of the synthesis procedure, specifically the base coupling efficiency, is the most significant factor influencing the final quality of an oligo. To obtain oligos which can be used without further purification, a high coupling efficiency during synthesis is essential. Oligos destined for further purification also benefit from a high coupling efficiency.
Synthesis efficiency is the most significant factor determining the final quality of an oligo even if the oligo is purified.
Proligo has developed and patented automated synthesizers and deprotectors (UFPS 24.1 UltraFast Parallel Synthesizer and UFPD 24.1 UltraFast Parallel Deprotector) incorporating technical innovations that permit a higher coupling efficiency than commercially available synthesizers.
Each individual step in the production of an oligo will influence its quality. The following information details each step in the production process, specifically:
- Synthesis
- Deprotection
- Desalting
- Purification
1) SYNTHESIS
The quality of oligo synthesis is largely determined by the coupling efficiency of each base. As oligo length increases, the number of incomplete synthesis products (species n-1, n-2,...) increases.
While coupling efficiencies of 98 to 98,5 % are possible with commercially available synthesizers, the Sigma-Proligo UFPS system typically exceeds a 99 % coupling efficiency. While this may be considered only a slight improvement, as seen in the table below, its significance becomes clear with the addition of each base:
For a coupling efficiency of
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The final purity for a 20 -mer oligo is
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The final purity for a 30 -mer oligo is
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The final purity for a 50 -mer oligo is
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2) DEPROTECTION
The deprotection step is crucial in the synthesis procedure. Additionally, it is particularly sensitive for labeled oligos as they are particularly susceptible to degradation during the deprotection step. To eliminate these problems, Proligo has developed its own proprietary technology to guarantee the stability of the oligo at this point in the synthesis. The success of the deprotection step is subsequently validated by a systematic quality control of each oligo.
3) DESALTING
Desalting entails the elimination of any remaining deprotection groups and trace solvents. Because salts can interfere with enzyme activity (such as Taq polymerase) and significantly reduce the intensity of the signal, desalting is a crucial step in synthesis. Desalting will also remove short synthesis products (< 10 mers), but will not remove nearly full length synthesis products (species n-1, n-2, n-3 which constitute the bulk of synthesis impurity) and as such should never be mistaken for a purification step. Only a sensitive purification procedure will practically eliminate these species, the concentration of which is dependent on the coupling efficiency during synthesis.
These are three principal methods by which desalting can be achieved:
- Precipitation
- Filtration (separation by size exclusion)
- Chromatographic techniques such as reverse-phase chromatography
Proligo UFP deprotector 24.1 technology is based on filtration or reverse-phase chromatography. This proprietary technique allows simultaneously deprotection and desalting.
4) PURIFICATION
The purification of an oligo is achieved by RPC (Reverse Phase Cartridge), PAGE (Poly Acrylamide Gel Electrophoresis) or RP-HPLC (Reverse Phase High Pressure Liquid chromatography). The purification technique is determined by the specific application and user preference.
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