I am currently learning about different types of vectors used in genetic engineering. While plasmids are widely used, I came across some references suggesting that inserts may sometimes be unstable in plasmids, especially when they are large or repetitive. Could anyone share which alternative vectors are considered more stable in such cases, and under what circumstances they are preferred over plasmids?
pJAZZ vectors are supposedly more stable than traditional cloning vectors for things that have high secondary structure or are repetitive, they're based on the unusual linear, non-integrating E. coli phage N15:
Article Linear plasmid vector for cloning of repetitive or unstable ...
That article provides some examples where they claim traditional cloning vectors do not work well, like long, extremely AT-rich regions and long regions of short nucleotide repeats.
In most cases though, I think unstable inserts are just cloned into circular vectors which contain plasmid origins of replication that are lowish copy, like p15A, or extremely low copy (1-2 copies per E. coli chromosome) like P1 and F plasmid based origins (sometimes called bacterial artificial chromosomes, or BACs). In the extremely low copy origins, they typically have to include partitioning functions on the plasmid to ensure even distribution to daughter cells after replication, so they aren't lost randomly. BACs were used pretty extensively for large sequencing projects prior to the widespread availability of long read sequencing specifically because they could easily carry large inserts (100+ kb) in a relatively stable fashion. The logic behind using a lower copy plasmid is to reduce the chances of intermolecular recombination between many copies of the same plasmid, make sure any replication/repair machinery isn't overwhelmed, or to reduce the fitness burden on E. coli so there's less of a selective advantage towards mutated plasmids.
Another common way is not to alter the plasmid itself but to alter the E. coli host carrying the plasmid. Long palindromes, such as those present in adeno-associated virus inverted terminal repeats, are sometimes unstable in plasmids, which can be at least partly alleviated by deletion of the sbcC gene:
Article Degradation and stable maintenance of adeno-associated virus...
The extremely common recA inactivating mutations present in most cloning E. coli strains were introduced to prevent recombination between repeats and to lessen the amount of multimers present in plasmid preps.
I will say however, that in my experience,the issue of large and/or repetitive DNA being unstable in plasmids is relatively rare. There seem to be a lot of people online who think that a 5 kb insert is "massive", meanwhile I just miniprepped a vector containing an entire 32 kb adenoviral genome inserted into a high copy plasmid with good yield and no mutations.
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