Gel electrophoresis is a molecular biology method used to analyze and separate DNA fragments based on their size. When you use gel electrophoresis to help you with molecular cloning, you will also need to be able to interpret and analyze the results of your gel.
For example, you may need to excise your digested plasmid DNA from agarose. However, when you look at your gel, you may see multiple bands in a given lane and wonder which one you should cut.
In this article, we will review the different forms of plasmid DNA and offer some useful tips to interpret your gel.
The Structure of Agarose
Agarose, produced from seaweed, is a polysaccharide. During polymerization, agarose polymers link non-covalently and form a network of bundles. This network consists of pores with molecular filtering properties.
Conceptual rendering of agarose gel at a microscopic level.
DNA separation occurs due to the mesh-like nature of the agarose gel. Smaller DNA fragments can move quickly through the pores, while larger fragments get caught and therefore travel slowly.
Let’s look at how DNA electrophoresis in an agarose gel works.
A well is a hollow pocket in the gel where the DNA is loaded. Because of the negatively charged phosphate backbone, DNA holds a slight negative charge that allows it to migrate to the positively charged anode. The travel distance of DNA molecules within an agarose gel is proportional to the log of its molecular weight.
How Does Circular Plasmid DNA Run During Gel Electrophoresis?
The gel electrophoresis conditions, including the presence of ethidium bromide (or alternative), gel concentrations, electric field strength, temperature, and ionic strength of the electrophoresis buffer, can affect the mobility of plasmid DNA.
The electrophoretic trapping is a balance between the electrophoretic force (pulling the circular plasmid DNA against the trap) and diffusion (allowing the circular plasmid DNA to escape a trap). So, large circular molecules have a greater chance to get trapped than smaller DNA forms.
Supercoiled DNA are more difficult to trap due to the small size of the twisted DNA.
4 Common Forms of Plasmid DNA
Covalently Closed Circle(CCC) Monomer
The covalently closed circular monomer is a negatively charged, supercoiled plasmid. Intact supercoiled plasmids have compact double-stranded DNA twisted around itself. Plasmid DNA isolated from bacterial hosts are usually present in this covalently closed circular form. Undigested plasmid DNA are usually supercoiled.
Open Circular (OC) Monomer
An open circular form is caused by the nicking (cleavage) of one DNA strand. UV irradiation or nucleases can cause this single-strand break. This structure is a relaxed and less compact form of plasmid. It also has less supercoiling than the covalently closed circular form.
The linear form is a result of a cleavage on both DNA strands caused by restriction endonucleases.
Open Circle (OC) Dimer, or "Concatemer"
An open circle (OC) dimer is an oligomeric form of a plasmid. This open circle dimer, or concatemer, can occur due to replication. Dimers are usually doubled in size compared to monomers.
How to Interpret Gel Electrophoresis Results
- In general, monomer supercoiled covalently closed circular forms move faster than any other forms because they have a compact supercoiled DNA structure. Therefore, they will appear further down in the gel.
- Open circular (OC) and linear monomers move slower than the supercoiled covalently closed circular monomer. They struggle to pass through the pores of the gel matrix than the covalently closed circular form. Therefore, open circular forms will appear higher in the gel. The order of migration is usually the supercoiled covalently closed circular monomer (the fastest), followed by the linear form and open circular form.
- Completely digested plasmid DNA usually shows up a single band on the gel, a linear form of the plasmid, in its lane. Undigested plasmid may have two forms show up in its lane: a covalently closed circular dimer and a covalently closed circular monomer. The dimer forms, due to their larger size compared to monomers, usually move slower than the monomers. Therefore, it will appear higher in a gel than a monomer. The covalently closed circular monomer form runs faster than the linear form of digested plasmid DNA.
Gel Electrophoresis Examples for Plasmid Forms. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. Lane 4: UV-irradiated plasmid DNA.
Now, as a practice, look at the agarose gel example below. Can you guess each plasmid form from these bands from the agarose gel below?
Gel Electrophoresis. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A.
For Lane 2, you may be able to see two bands. The faint band on top is the open circular form and the one below it is the supercoiled covalently closed circular form. Remember, the supercoiled covalently closed circle is more compact than open circle and can travel further during a given time.
For the lane 3, it’s the completely digested plasmid, so the band you see is a linear form.
Tips To Identify The Bands In Your Agarose Gel
During gel electrophoresis, you may have to load uncut plasmid DNA, digested DNA fragment, PCR products, or genomic DNA into the wells. The next step is to identify those bands. For that, we summarize what we have described in this article and quick tips to help with identification.
- Uncut plasmid DNA on the agarose gel is easy to identify because it may have two forms of plasmid (OC and CCC forms).
- Digested plasmids, digested DNA fragments, PCR products, and genomic DNA may all have one single band. To identify these bands, you will have to check on their size by consulting the DNA ladder. Your digested plasmid has a linear form with the size in between open circle and supercoiled covalently closed circular forms of the uncut plasmid. Genomic DNA will be a larger size. So, genomic DNA usually shows up at the very top of your gel (very close to your well).
- Digested DNA fragments may have a single band at almost a similar size as your PCR product.
At the bottom of the PCR product lane, you may see a faint band indicating small molecules. These small molecules are your primer molecules that link to other primer molecules to form a primer dimer.
Gel Electrophoresis. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. Lane 4: Digested PCR product (or DNA Fragment). Lane 5: PCR Product (with a faint primer dimer band). Lane 6: Genomic DNA.
To learn more about how to interpret DNA gel electrophoresis, watch our video below:
Agarose LE (Molecular Biology Grade) (Catalog No. A-201)
Low Melt Agarose (Catalog No. A-204)
1 kb DNA Ladder (Catalog No. D010)
1 kb PLUS™ DNA Ladder (Catalog No. D011)
100 bp DNA Ladder (Catalog No. D001)
100 bp PLUS™ DNA Ladder (Catalog No. D003)
50 bp DNA Ladder (Catalog No. D100)
VersaLadder™, 100-10,000 bp (Catalog No. D012)
Gel Loading Dye Products
6X Blue Loading Dye (Catalog No. L002)
6X Green Loading Dye (Catalog No. L001)
Cole, K. D., & Tellez, C. M. (2002). Separation of large circular DNA by electrophoresis in agarose gels. Biotechnology progress, 18(1), 82-87.
Green, M. R., & Sambrook, J. (2019a). Agarose gel electrophoresis. Cold Spring Harbor Protocols, 2019(1), pdb. prot100404.
Johnson, P. H., & Grossman, L. I. (1977). Electrophoresis of DNA in agarose gels. Optimizing separations of conformational isomers of double-and single-stranded DNAs. Biochemistry, 16(19), 4217-4225.
Schleef, M. (2008). Plasmids for therapy and vaccination: John Wiley & Sons.
Schmidt, T., Friehs, K., & Flaschel, E. (2001). Structures of plasmid DNA. Plasmids for therapy and vaccination, 29-43