Do I need to have my oligonucleotides purified?

The decision to have your oligonucleotides purified should be based on several factors including:

  • the application in which you will be using the oligo
  • the length of the oligo
  • whether the oligo contains any modifications
Oligonucleotides are synthesized base by base using chemical reactions that are only ~99% efficient. So with each base addition, ~1% of the oligos will not pick up the proper base. We try to prevent these oligos from participating in later base additions by running them through a capping reaction. Most of these oligos will pick up this capping reagent and will become truncation mutants. Unfortunately, because the capping reaction is not 100% efficient, some oligos will remain uncapped and able to react with later bases. This leads to the formation of deletion mutants; the deletion can occur anywhere within the sequence. The resulting oligonucleotide synthesis reaction is thus a mixture of full length oligos, truncated products, and oligos with internal deletions.

Part of your decision to request (additional) purification should be based on how sensitive your particular application is to the presence of truncated products or those with internal deletions.

PCR or sequencing:
standard desalting is perfectly sufficient as the truncations and deletions will not affect your results appreciably.

Cloning, mutagenesis, and gel shift: For these applications, full length product is of utmost importance, and PAGE purification is strongly recommended. PAGE purification results in the highest purity level in terms of full length product -- routinely achieving 85% full length product.

PAGE purification does tend to result in lower yields than HPLC purification, however.  If you need a relatively clean product, but also need a high yield, you should consider standard HPLC for oligos up to 50 nt, and IE-HPLC for longer oligos.

Modified Oligos: You should also take the types of modifications on your oligo into account when choosing a purification method. PAGE purification can damage certain modifications, including many fluorophores and  some modifications used for attachment. PAGE purification should be avoided for the following modifications: any fluorophore, acrydite, amino modifiers,  biotin, digoxigenin, I-Linker, Spacer 18, and thiol modifiers. HPLC or IE-HPLC would be the purification of choice in these cases.

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