• Oligonucleotide Purification: Basic Information and Different Methods

    Oligonucleotide is a small nucleic acid polymer that is either formed by bond cleavage or synthesis. It is basically made up of a sequence of nucleotide residues that comprise the entire molecule. Oligonucleotides can easily bind with their complementary DNA or RNA – the reason why they are very important for detecting DNA and RNA especially in procedures like DNA microarrays, southern blots, ASO analysis, Fluorescent in Situ Hybridation (FISH), and artificial gene synthesis. Oligonucleotides are also used in antisense gene therapy.

    Oligonucleotides need to be purified after DNA synthesis depending on the probable degree of contamination in order to avoid undesired effects on intended experiments. Purification will separate the full length oligonucleotide from the truncated sequences. However, some processes do not need to purify oligonucleotides especially if the degree of contamination is less.

    Oligonucleotides purification performs three basic functions:

    1. Separate full-length oligonucleotides from incomplete products
    2. Remove modified oligonucleotides from incomplete deprotection, depuniration, and demerization.
    3. Desalt oligonucleotides and remove the cleaved blocking groups

    The methods used to purify Oligonucleotides may vary depending on the factors listed below.

    1. Size of the oligonucleotides
    2. Degree of purity required
    3. Quantity to be purified
    4. Availability of instruments
    5. Time available
    6. Number of samples to be purified
    7. Cost of the method

    Listed below are the different methods used to purify oligonucleotides.

    Gel Filtration

    This is the simplest method of oligonucleotides purification which involves the separation of components based on the size of molecules involved. Using the gel filtration column, scientists retain the cleaved protecting groups and short truncated sequence from the gel matrix and elute the larger Oligonucleotides molecules.  Scientists also use this method to remove salts from the Oligonucleotides; however it is not preferable for smaller size molecules.

    High- performance Liquid Chromatography (HPLC)

    This method is used to separate oligomers with similar size and is preferable for the purification of synthetic oligonucleotides. The HPLC system is composed of several  computer- operated components  including  an injector, pumps for delivery of solvents through the column, an interchangeable HPLC column, a column oven, a solvent mixing system, and a detector (normally UV/visible).

    Reversed-Phase HPLC

    This method is used to separate oligonucleotide and contaminants based on varying hydrophobicity. Some samples that are suited for this method may include any strands of oligonucleotide that have not been completely deprotected, modified oligonucleotides like fluorescent dyes, and short oligonucleotides.

    Anion-exchange HPLC

    This method is used to purify oligonucleotides that have different electrical charges by using a salt-gradient solution.  The resolution of Anion-exchange HPLC is best for smaller quantity purification; however it can also be used to purify hydrophobic oligonucleotides by adding organic solvents to the solvent system.


    Although the yield for this method is lower compared to other methods, this method is highly recommended for highly purified results. PAGE purifies oligonucleotides depending on their molecular weight resulting to excellent size resolution and a 95% – 99% purity levels.


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