Understanding Plasmid Multimers

Dec 12, 2023

Plasmids are small, circular, double-stranded DNA molecules found in bacteria and some other single-celled organisms like archaea. They exist separate from the chromosomal DNA and carry genes that are often advantageous but not essential for the host cell's survival.

Purified plasmids serve as versatile tools in scientific research and practical applications. In research laboratories, they're pivotal for gene cloning, allowing scientists to deliver specific genes for functional studies. In pharmaceutical industries, plasmids play a crucial role in recombinant DNA technology, enabling the production of therapeutic proteins and facilitating gene therapy by delivering modified genes into cells to treat genetic disorders.

When handling plasmids, many researchers will come across a naturally occurring phenomenon called multimerization. In this blog, we will explore plasmid multimerization—unraveling the complexities surrounding their formation, the biological underpinnings driving their existence, and the ramifications they impose on genetic studies and biotechnological applications.

Understanding Plasmid Multimerization

At its core, plasmid multimerization refers to the occurrence where two or more identical or similar plasmids become linked or concatenated, resulting in the formation of larger plasmids with repeated sequences¹. This process occurs through recombination events between homologous regions of 2 or more molecules. The resulting multimers can range in size and complexity, depending on the number of plasmid copies involved in the concatenation and the specific regions of recombination.

Existing molecular machinery in the cell is able to resolve the formation of plasmid multimers, however, when cellular enzymes fail to execute during cell division, multimers persist in the system.

Implications of Plasmid Multimerization

Plasmid multimerization introduces structural variations, transforming the typical circular plasmid into larger, more complex structures. In principle, so long as the essential plasmid elements remain unaltered, plasmid multimers (dimers, trimers, tetramers, etc.) operate with similar efficiency and effectiveness as their monomeric counterparts.

There are concerns that the presence of multimers can impact factors such as plasmid stability, cellular growth rates, and transfection/transformation efficiency, but studies are ongoing to confirm these claims.

It was originally hypothesized that plasmid dimerization had a severe effect on stability since it is 2x more likely to replicate than a plasmid monomer². This would theoretically lead to the rapid accumulation in a growing cell line, resulting monomeric plasmid loss. A study by Summers et. al., found that dimer-only cells grow 10% slower than monomer-only cells, stabilizing the population and reducing the proportion of dimers. In fact, it was reported that only 4.5% of the total plasmids were dimers. This slower growth rate was seen in both in-vitro and in-vivo experiments, showing that plasmid instability resulting from multimerization alone, is relatively low³.

It should be noted that multimers may also have a lower transfection and transcription efficiency due to their large size and ability to be easily trafficked into the cell⁴. However, it is unlikely that lowered efficiencies from a few dimers will have larger downstream effects due to their low proportion within the plasmid population. Researchers experiencing decreased efficiency should explore other factors beyond dimerization, such as cell line characteristics, transfection methods, or other aspects influencing gene expression.

Detecting Plasmid Multimers

There are several methods to detect plasmid multimers in experiments but the most common are:

• Agarose Gel Electrophoresis: Running undigested plasmid DNA samples on an agarose gel allows for the visualization of different-sized DNA molecules. Plasmid multimers typically appear as higher molecular weight bands compared to monomeric plasmids, showing up as distinct bands on the gel.
• Long-read Sequencing: Long-read sequencing of plasmid DNA can reveal variations in sequence lengths that are indicative multimers.

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Citations:

1. Bedbrook JR, Ausubel FM. Recombination between bacterial plasmids leading to the formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: 797459.
2. Summers DK, Beton CW, Withers HL. Multicopy plasmid instability: the dimer catastrophe hypothesis. Mol Microbiol. 1993 Jun;8(6):1031-8. doi: 10.1111/j.1365-2958.1993.tb01648.x. PMID: 8361350.
3. Field CM, Summers DK. Multicopy plasmid stability: revisiting the dimer catastrophe. J Theor Biol. 2011 Dec 21;291:119-27. doi: 10.1016/j.jtbi.2011.09.006. Epub 2011 Sep 19. PMID: 21945338.
4. Jobling MG. Plasmid multimer status and not DNA topology likely affects luciferase assay reproducibility. Biotechniques. 2020 Sep;69(3):156-157. doi: 10.2144/btn-2020-0009. Epub 2020 Jun 12. PMID: 32527156.