Do I need to order purified oligonucleotide for cloning experiment?
There are several possible explanations for apparent errors in the sequence of oligonucleotides used in cloning experiments. Potential problems associated with using the beta-cyanoethyl phosphoramidite chemistry in oligonucleotide synthesis are:
- The G base may have been converted to the enol tautomer, 2,6 diaminopurine, recognized as A by DNA polymerase. Thus, clones generated from an oligo with this modified base will appear as a G to A transition.
- The chemical process of synthesis may cause depurination. Depurinated oligos can degrade at the deprotection stage, but a small percentage can remain. Clones containing an inserted oligo that was depurinated will appear as having an A or G deletion. Oligos that are purine-rich have an increased risk of having these artifacts.
- Usually, failed sequences that do not couple to the next incoming base are capped to prevent further synthesis. Unfortunately, sequence failures that are not capped are still capable of further synthesis and will appear as deletions in cloning experiments. These events are usually rare, and the effects can be overcome by screening additional clones for those that contain an oligo with the correct sequence.
- During the base addition step, a small percentage of the incoming bases may couple with each other before coupling with the growing oligo chain. Clones generated using these oligos will appear as an insertion. Again, this event is rare, and the effects can be overcome by screening additional clones for those oligos with the correct sequence.
- Therefore, it is highly recommended that HPLC or PAGE purified oligos be used for cloning experiments and that more than one clone should be collected and screened.