Is oligonucleotide purification necessary?

How many impurities are present in an unpurified oligonucleotide?

Oligonucleotides are synthesized by the phosphoramidite method through sequential extension of the chain with activated monomers. Target purity depends on coupling efficiency at every synthesis step. With an average coupling efficiency of 98.5%, the theoretical yield for an oligonucleotide of N residues is (0.985)^N-1×100%. The longer the oligonucleotide, the higher the proportion of truncated products: the minimum final yield is about 81% for a 15-mer and about 60% for a 35-mer.

The longer the oligonucleotide, the more important purification becomes.

What purity is required?

Coupling is not quantitative, so shorter products accumulate together with the target sequence. Purification removes these truncated sequences and isolates the target-length oligonucleotide. SYNTOL uses gel electrophoresis or HPLC, with conditions selected for sequence, length, and oligonucleotide type. The stated source material gives a typical guaranteed purity range of 90–98% for ordered oligonucleotides.

Although unpurified primers may be used for PCR, the source article notes that specificity and sensitivity can decrease and that unpurified primers degrade faster during storage.

Purification by PAGE

Polyacrylamide gel electrophoresis (PAGE) is widely used for analytical and preparative purification. The main stages are sample preparation, electrophoresis, visualization and isolation of the target product. Separation depends on molecular weight and oligonucleotide length. Preparative purification is performed in 10–20% polyacrylamide gel, depending on oligonucleotide length, under denaturing conditions with 7 M urea. Length markers allow the target-length band to be identified.

The gel is visualized on a TLC plate under a UV lamp; quantities above 0.1 optical density units can be distinguished. The target band is excised, recovered and desalted. Concentration is measured at 260 nm. PAGE can provide 95–99% purity for short oligonucleotides (<25 residues) and 85–90% for long oligonucleotides (>30 residues). The source article limits this approach to relatively small quantities (up to 10 optical density units) and notes reduced efficiency for G-rich sequences that form stable secondary structures.

Purification by reverse-phase HPLC

At the final stage of automated synthesis, the target oligonucleotide carries a hydrophobic 5′ dimethoxytrityl group. Reverse-phase HPLC separates this product from shorter impurities that do not carry the group. More hydrophobic molecules are retained longer on the column during gradient elution. After isolation, the dimethoxytrityl group is removed and the oligonucleotide is desalted; purity is then checked by analytical gel electrophoresis.

Reverse-phase HPLC allows larger quantities to be purified, typically to 90–95% according to the source article. Separation efficiency may decrease for oligonucleotides longer than 50 residues, and G-rich oligonucleotides with highly stable secondary structures are not suitable for this method.