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Purification – When Should I purify?
For a diagnostic assay, you should purify when failure sequences, e.g. n-1, n-2 etc., influence the outcome of the reaction either by interfering or lowering the sensitivity of the intended assay. For oligos that have 5′ or 3′ dye modifications, assay sensitivity may be diminished due to competing full-length oligos on which the dye is not attached.Purification is required when assay c.v.’s improve due to more consistent purity.
For oligos intended as therapeutics it’s mandatory that you control the level of impurities and therefore purification and characterization of the impurities is required.
What purification methods do you offer?
We offer both Reverse Phase and Ion Exchange HPLC (IEX).
Reverse Phase HPLC is the method of choice when separation can be achieved by the relative hydrophobicity of the oligo. This is typically the case when 5′ and 3′ modifiers are used such as dyes. Reverse phase chromatography is not effective at removing the n-1 failure sequences.
Ion Exchange HPLC is the method of choice when separation by charge provides maximum separation, such as unmodified oligos. IEX HPLC is also the method of choice for oligos greater than 50 bases.
For difficult sequences we may employ both techniques in order to acheive the high standards you require.
Methods for Purification of Synthetic Oligonucleotides
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