PCR amplicons are commonly used for cloning. Primers made from synthetic oligonucleotides often contain a small percentage of incorrect sequences which, combined with ligation and cloning errors, lead to a small percentage of clones that contain random, incorrect bases. Occasionally, researchers observe the same error repeated in many or all of their clones. This is usually a single-base deletion near the ligation site. The precise mechanism for these errors is unknown. However, in most cases, the issue appears to be unrelated to oligo synthesis based on the following evidence:
- Mass spec data confirming the accurate oligo mass prior to cloning indicate that the majority of the product is of the correct mass and free from deletions.
- The deletions are seen only in cloning applications and not in other cell-free, sequence-sensitive applications, such as next-generation sequencing.
- Deletions are sometimes reduced or eliminated when the same amplicon is cloned using a less recombinogenic cell line.
- Deletions are sometimes reduced or eliminated by changing the vector used for cloning or by moving the cloning site within the vector.