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Technical Bulletin (Adobe Acrobat)
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When ordering their oligos, many people wonder how pure is pure enough. This technical bulletin will help you select the best purification option for your oligo, depending upon the oligo type and its intended application.
In DNA synthesis, each nucleotide is coupled sequentially to the growing chain. In each coupling cycle, a small percentage of the oligo chains will not be extended, resulting in a mixture of full length product and truncated sequences.
After the oligo is cleaved from the support and the protecting groups are removed, purification can separate the full length product from the truncated sequences. In general, the purity required for a specific application depends on the potential problems from the presence of truncated oligomers. For some applications, it is crucial that only the full length (n) oligo be present. For others, the presence of shorter oligos (n-1,n-2,...) will not affect the experimental results.
Purification of Modified Oligos
Oligos that have been modified with biotin, fluorescein, 6-FAM, HEX, and TET can be purified by any of the methods listed below. For oligos that have been modified with digoxigenin, the ABI dyes, Texas Red, and any Molecular Probes dyes, we recommend purification by HPLC or PAGE. 5' Phosphorylated oligos cannot be purified with RP1 or HPLC. Therefore, PAGE is a suitable alternative.
Desalting
At Genosys, every oligo is desalted free of charge. Desalting removes residual by-products from the synthesis, cleavage, and deprotection procedures. For many applications (including PCR), desalting is fine for oligos <= 30 bases; the overwhelming abundance of full length oligo outweighs any contributions from shorter oligos. For all oligos>30 bases in length, it is recommended that an additional method of purification be considered.
Reverse-phase cartridge purification (RP1)
Separation on a reverse-phase cartridge offers the next level of purity (typically 80-90%). The level of purity for RP1 is almost equivalent to that provided by HPLC and the recovery is often higher. The basis of the separation is the difference in hydrophobicity between full length product (which contains a 5'-DMT) and truncated sequences (without DMT groups). Because the differences in hydrophobicity between the full length-DMT product and non-DMT truncated sequences are reduced as the oligo length is increased, cartridge purification is not recommended for oligos > 50 bases. For most high resolution applications, cartridge purification will provide the necessary purity level.
HPLC Reverse-phase
Reverse-phase HPLC operates on the same principle as the reverse-phase cartridges, but typical yields a product of 90-97% purity. The capacity and resolving properties of HPLC columns are also much greater than cartridge devices, so HPLC is the method of choice for purifying larger quantities of oligos (i.e. >= 1 umol). As with cartridges, reverse-phase HPLC is usually not recommended for purifying oligos longer than 50 bases.
PAGE
With the excellent resolution of PAGE, a 95-99% purity can be achieved. The basis of the separation is charge and MW. In most cases , full length (n) oligo can be separated from oligos only one base shorter (n-1). PAGE is the recommended technique for purifying oligos >50 bases long. Yields from PAGE are lower than from other methods due to the relative inefficient extraction of oligos from the gel.
Gel Filtration
For antisense and triple helix studies, all S-oligos receive a final desalting by gel filtration. Gel filtration is also recommended for any oligo to be used in cell culture or in vivo studies. Gel filtration prevents cytotoxic effects from trace synthesis by-products or trace solvents which may carry over from purification.
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