Comparison of oligos purification methods

Â Â Â Â In order to purify the synthesized oligonucleotide, a method of performing oligos purification by desalting, BioRP , PAGE , HPLC, or the like is employed. The focus of each method is different, and the most suitable purification method should be selected according to the requirements of the specific experiment:

Desalting Â Â Â After the oligonucleotide is synthesized, in order to purify the oligonucleotide to become a natural DNA structure, it is first necessary to remove the protecting group. The concentrated oligonucleotide is separated from the solid phase carrier by concentrated ammonia treatment, the protective group of 2- cyanoethoxy - phosphodiester bond, and the protecting group of the base (benzoyl and isobutyl) The base is removed to form a natural DNA structure. However, after the necessary deprotection step is completed, the oligonucleotide mixture also contains several small molecule organic compounds that must be removed. The process by which all non-essential organic compounds are removed is often referred to as desalting. Desalting purification can be carried out by means of a reverse silica gel column. Although desalting purification removes all non-essential organic impurities, it does not effectively remove traces of prematurely terminated oligonucleotide miscellaneous strands in the synthesis. However, desalted oligonucleotides are still able to satisfy basic biological studies such as PCR detection.

BioRP If the oligonucleotide is synthesized in the form of " trityl " , the N -methyl oligonucleotide contains 5' -DMT group, the early terminated oligonucleotide does not contain this group. Because the DMT base has strong lipophilic properties, there are 5' The oligonucleotide of the -DMT group has affinity with the reverse phase resin, and thus the reverse phase affinity resin is generally used for the purification of the oligonucleotide. Using reverse resin and 5' - DMT oligonucleotides have strong affinity, but in advance The terminated oligonucleotide does not contain a DMT group, so we were able to successfully separate the desired N- methyl oligonucleotide from the pre-terminated oligonucleotide impurities.

PAGE For the purification of long oligonucleotides ( 50-100 mer ), we recommend PAGE purification, which uses a cross-linked polyacrylamide gel ( electrophoresis ) as a purification matrix. Although PAGE shows high purification efficiency (> 98% ), it has some drawbacks in terms of additional steps, such as extraction and desalting after PAGE , which in turn leads to a decrease in purification yield.

HPLC Â If the synthetic oligonucleotide is applied to cloning, targeted mutagenesis or quantitative gene detection, its purity requirements are high. Desalting or OPC- purified oligonucleotides may not meet the requirements, and HPLC purification is widely used for this purpose. As a purification resin, an anion exchange resin or a reverse resin is used for oligonucleotide purification. HPLC of anion exchange resins generally shows a purity efficiency of 95-98% , which is sufficient for purification of the 35-mer oligonucleotide. Reversed resinated HPLC exhibited similar purification efficiencies to the HPLC of the anion exchange resin . Since the purification efficiency of HPLC largely depends on the length of the oligonucleotide, long oligonucleotides (greater than 35-mer) cannot be efficiently purified by HPLC .

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