Mutations or alternations in sequence are rare in synthetic oligos but they may happen. Oligos may become depurinated in some positions during the synthesis process. The longer the oligonucleotide, the more depurinated sites may occur. These depurinations may become visible in applications like cloning where single molecules are selected and propagated. Typically the depurinated sites are replaced by any base. Usually only some oligonucleotide molecules are affected. If you have sequenced only one clone and found a 'mutation' in it, sequencing another independent clone will in most cases obtain the correct sequence. When choosing clones, it is the most important to select independent ones. To get independent clones, the pre-incubation time before plating should be based on the manufacturer instructions of your cloning system. Optimal density of the clones on the plate ensures that well separated clones can be selected. Longer oligos used for cloning should be purified to remove any truncated sequences. As the risk of depurinations is increasing with the longer oligonucleotide, we recommend to select sequences as short as possible. If long oligos (>40nt) have been used for cloning, consider sequencing more clones. Please note that synthetic oligos do not contain a phosphate at the 5’end which is necessary for enzymatic ligation reactions. The phosphate can be added as a modification to the 5’ end of the oligo.
Articles in this section
- Why should oligonucleotides be purified?
- Why does MALDI analysis of my oligos containing one or more Fluorescein-dTs give an incorrect mass even though they give only a single, fluorescent band on a PAGE gel?
- What shall I do with the RNA synthesis product from your company?
- What exactly is an OD?
- Sometimes I notice that the dried oligos have a brown color after drying. Is this the natural color of DNA or is the brown color a sign of contamination?
- I used oligos for cloning. When I sequenced a clone and found a mutation within the oligo sequence. Can mutations happen in synthetic oligos?
- How to store the synthetic Oligos?
- How should I resuspend my fluorescent dye-labeled oligos?
- How long will my fluorescent dye-labeled oligos last?
- How do you calculate the extinction coefficient of an oligo?