Linear Plasmid DNA

BioInnovatise Plasmid DNA Team

Updated September 16, 2024

Plasmid DNA is naturally circular, especially in bacterial cells such as E. Coli where is easily manipulated for molecular cloning or mutagenesis. The transition from supercoiled circular plasmid DNA to linear plasmid DNA can occur through several mechanisms:

Enzymatic digestion via restriction enzymes
Mechanical shearing
UV radiation and chemical damage
Topoisomerase action
Homologous recombination

How Does Linear and Circular Plasmid DNA Behave Differently?

Once linearized, a DNA construct behaves differently than circular plasmid DNA in electrophoresis and in its interactions with cellular machinery. But how exactly? Linear plasmid DNA and circular plasmid DNA have their separate advantages for different applications. Here is an overview of how each of them behaves during assay tests and interacts within cells.

1. Electrophoresis Mobility

In gel electrophoresis, circular supercoiled plasmid DNA migrates faster through the gel than linear DNA. Supercoiled DNA is more compacted and moves easily through the pores of the gel.
Linear DNA migrates more slowly compared to supercoiled plasmid because it is less compact. If the plasmid is relaxed (nicked circular DNA), it will migrate even slower than linear DNA.

2. Transformation Efficiency

Circular, highly supercoiled plasmids are more efficient at being taken up by bacterial cells during transformation. The compact structure of supercoiled DNA allows it to pass through the bacterial membrane more easily.
Linear constructs have much lower transformation efficiency because bacterial cells often treat linear DNA as foreign and degrade it using exonucleases

3. Stability

Circular plasmid DNA is more stable in both cells and in vitro, particularly in its supercoiled form, which protects it from degradation by nucleases.
Linear plasmid DNA is more prone to degradation by exonucleases, as its exposed ends are vulnerable to enzymatic attack. Cells have exonucleases that recognize and degrade linear DNA.

4. Cloning and Recombinant DNA Techniques

Circular plasmids are often the default structure for cloning, where they can replicate independently inside bacterial cells.
Linear Plasmid: Linear plasmids are less suited for replication unless they are joined back together or integrated into another DNA molecule. In some cases, linear plasmids are used in specialized cloning techniques, but they are typically modified to circularize in the host cell.

5. Replication

Circular plasmids, especially in bacteria, are able to replicate autonomously due to the presence of an origin of replication.
Linear plasmids often require additional sequences, such as telomere-like structures in some organisms, to replicate effectively. If they don’t have these sequences, they typically won’t replicate in bacteria or other cells.

6. Integration into Genomes

Circular plasmids can integrate into the host genome but usually require recombination events.
Linear plasmids are more readily incorporated into genomes through homologous recombination because the open ends can facilitate easier integration into double-strand breaks within the genome.

Can My Plasmid Construct Become Circular Again?

Yes! Linear DNA can become circular again through various molecular processes that our plasmid DNA team can help with including restoration techniques with DNA ligase enzymes, homologous recombination during transformation, NHEJ, and T4 DNA ligase processes.

If you are interested in having your construct , just let our team know and we would be ahppy to help with that.

Request Molecular Cloning or Mutagenesis Productions On Linear Constructs

Linear constructs can indeed be used for molecular cloning and mutagenesis productions if you require improvements to your construct, require a sequence be changed, or are looking to create a plasmid DNA library. Just make sure you include the information when you start a production.

If you are concerned that you do not have enough linear plasmid DNA for your research applications, we can produce higher volumes via plasmid midiprep, plasmid maxiprep, or large scale productions (> 1 gram). See our plasmid DNA preparation services.

The diagram above illustrates the plasmid prep production process for linear endotoxin free and animal component free plasmid DNA.

Can I Package A Linear Plasmid DNA Construct Into A Viral Vector?

Sure! Linear plasmid DNA, just like supercoiled circular plasmid DNA, can be effectively and efficiently packaged into viral vectors such as lentivirus and retrovirus however the efficiency of packaging and delivery depends on the viral vector system, the size of the DNA insert, and the target cells or tissues.

Our plasmid DNA team is able to work with your linear plasmid DNA construct when it comes to molecular cloning, mutagenesis, and viral vector packaging.

Precision medicine research and development progresses everyday, and with it, the need for high-integrity plasmid DNA.

Want to learn more about the latest in plasmid research? Our colleagues at Science Direct and the American Society for Biochemistry and Molecular Biology are always collecting and publishing the latest information and research.

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