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.
Observations from Cloning
While there are no clear rules that predict when and where such errors will occur, the deletions tend to occur near ligation sites in areas of high secondary structure, particularly at the ends of predicted hairpins that close loop structures, and at the termini of short sequences identical to those in the E. coli genome. However, these may not be the only regions that produce deletions. We have also observed that two identical plasmid vectors containing nearly identical sequences may behave differently; one may generate the required clone while the other consistently generates clones containing deletions.
Resolving Cloning Deletions
There are steps you can take to increase the odds of generating correct clones.
- Before changing your cloning protocol, make sure you have sequenced enough clones. In many cases, even though a small number of clones contain the wrong sequence, a proportion of the clones may be correct. If sequencing additional clones does not solve the problem, try changing one or more of the cloning products used in your protocol. We recommend altering the competent cells and/or the plasmid vectors.
- Use a competent cell line developed to limit recombination and to support the growth of unstable sequences. These cell lines limit errors that may be introduced when some sequence motifs are transformed into other bacterial cells.
- Use vectors that are designed to facilitate cloning of unstable DNA sequences, e.g., small plasmids designed to reduce secondary structures and some low-copy vectors.
- If it is not possible to change vectors, site-directed mutagenesis can frequently correct the sequence error.
While these suggestions do not guarantee that cloning will be successful, they can improve the success rate and may even speed up cloning by addressing the correct source of the deletions.