Gel electrophoresis uses electricity to separate fragments of DNA based on their length. An understanding of how DNA migrates in an electrical field is needed in order to properly interpret the result of a gel electrophoresis run.
The negative charge on the sugar-phosphate backbone of DNA polymers cause them to migrate towards the positive electrode when placed in an electrical field. The rate of movement towards the positive end of the electrical field is influenced by the composition of the material the DNA is placed in.
For gel electrophoresis, DNA is placed in a porous gel. The pores restrict the movement of the DNA and creates an environment in which each individual DNA fragment’s rate of movement varies based on its length. See the gel electrophoresis overview illustration for more on the components used in gel electrophoresis.
The following illustration shows migration patterns in a gel when DNA of different lengths are loaded into a gel. The tubes on the right contain DNA samples and standards. Drag samples from the tubes to the wells to begin.
The starting point for analyzing DNA samples using gel electrophoresis requires a number of things including:
- A gel in a gel box with the wells oriented towards the negative electrode
- A set of samples and standards mixed with loading dye*
The samples can contain DNA fragments of known or unknown length. Standards (or DNA ladders) are run on the gel in order to get a better estimate of the lengths of the DNA fragments in the samples. These standards can be prepared in the lab ahead of time or purchased pre-made.
The loading dye present in both the samples and standards helps make sure each well loads properly and makes it easy to keep track of which wells already contain DNA.
Once the wells are loaded, the power is turned on. The current creates the electrical field across the gel needed to force the DNA towards the positive end of the circuit.
At the beginning of the run, DNA of all lengths are relatively close together. As time goes on the diference in the rate of migration of fragments of different length causes them to separate.
Longer fragments take more time to move through the pores in the gel so they move more slowly. Each individual strand of DNA in a sample is too small to be seen. Gel electrophoresis works because the samples and standards contain billions of copies of the DNA fragments being analyzed. The movement of all of these billions of fragments of the same lengths moving together forms the visible bands.
DNA at the same vertical position in two different lanes are fragments of the same length. By comparing the position of each band to bands in the standard or ladder the lengths of bands in the samples can be estimated.
Standards need to be run in each gel because the absolute* position of a band may vary from run to run even though the relationship between bands doesn’t change: shorter fragments move faster and end up lower in the gel when compared to longer fragments.
Even with billions of copies of a DNA fragment at a position in the gel, the DNA is not visible until it is stained or marked in some other way. Visualizing the DNA is done after the power is turned off using one of a number of different DNA stains available for this purpose.
Test your understanding of these concepts with the band migration practice problems
- Problem Sets