In this lesson, you will learn about the role that ethidium bromide, loading buffers, and DNA ladders play in visualizing DNA and determining the size of DNA fragments in agarose gel electrophoresis.
Gel Electrophoresis Review
Agarose gel electrophoresis plays a critical role in analyzing DNA in laboratory experiments. It is a method of separating biological molecules using an electrical current. We learned previously that DNA molecules can worm their way through the pores in an agarose gel. The negative charge of DNA molecules propels them through the electrical field toward the positive electrode. Shorter DNA molecules can travel farther in an agarose gel in a given amount of time than longer counterparts. Agarose gel electrophoresis provides a means of analyzing DNA by separating molecules based on size. However, in and of itself, agarose gel electrophoresis does not provide a means of visualizing the DNA. That role is played by two dyes: ethidium bromide and loading buffer.
Ethidium bromide binds to DNA and fluoresces under an ultraviolet light
Ethidium bromide is a molecule commonly used to visualize DNA in agarose gel electrophoresis experiments. It both binds to DNA and fluoresces under the proper conditions. Ethidium bromide is known as an intercalating agent. The flat structure of ethidium bromide allows it to intercalate, or insert, between nitrogenous bases of a DNA molecule. This interaction is especially useful because of ethidium bromide's second characteristic. When it is exposed to ultraviolet light, ethidium bromide fluoresces. Thus, this chemical provides both a means of tagging DNA molecules and a means of visualizing them.
However, note that any molecule that binds to or in any way alters the molecular structure of DNA can be dangerous to a living organism. Although there is some debate about the exact nature of the health risk posed by exposure to ethidium bromide, most organizations and agencies do consider it a risk. Because of the intercalating characteristic of ethidium bromide, it is believed by many to pose a mutagenic risk. However, with strong safety protocols in place, ethidium bromide continues to be one of the most common methods of visualizing nucleic acids in agarose gel electrophoresis experiments.
Well, that's the end of the story, right? Agarose is going to separate DNA molecules based on size, and ethidium bromide is going to tag the molecules so we can visualize them under UV light. Well, not exactly. DNA, like many biological molecules, is colorless. To complete the electrical circuit, the gel must be bathed in a salt solution called electrophoresis buffer. Water is also colorless. See the problem?
Loading buffer is a solution added to a DNA sample to make it visible to the naked eye
If DNA is colorless and the electrophoresis buffer bathing the agarose gel is also colorless, how are we going to tell if we've inserted the DNA sample into the well of the gel or not? We could add ethidium bromide and perform the entire protocol under UV light, but that seems unnecessarily dangerous to our health. The answer is that we need to use a different solution, called loading buffer. Loading buffer is added to a DNA sample to give it color to the naked eye.
Notice that monitoring the progression of the dye molecules in the gel will also allow us to determine when the protocol is complete. Fantastic. Now I can tell if I've accurately inserted our DNA sample into the well. But there's still one more problem. DNA is an aqueous molecule.
That means that it is hydrophilic and will dissolve in water. If our DNA samples dissolve in the water of the electrophoresis buffer, the DNA molecules will be spread evenly throughout the solution and will not stay localized to the given well.
So loading buffer provides one more function in gel electrophoresis. Loading buffer also increases the density of the sample. Recall that denser objects sink, so adding loading buffer to the DNA samples will enable the DNA molecules to sink into the wells in the gel in preparation for gel electrophoresis.
While qualitative visualization of DNA molecules is certainly helpful, experiments often require much more precise analysis of DNA fragment size. Rather than only being able to conclude that fragment one is larger than fragment two, scientists often need to be able to conclude that fragment one is three kilobases and fragment two is one kilobase. To be able to determine the size of a DNA fragment, scientists use something called a DNA ladder. A DNA ladder is a solution composed of DNA fragments of known length.
A DNA ladder helps scientists to determine the size of a DNA fragment
Since the size of each band in a DNA ladder solution is known, they can be used to determine the size of DNA fragments in experimental samples. Consider the following experiment. A four-kilobase piece of linear DNA has been digested with a restriction enzyme. A DNA ladder, uncut DNA, and digested DNA samples have been run on an agarose gel. See how the bands of the DNA ladder provide a point of comparison for the experimental samples? Since we know the sizes of the DNA fragments in each band, we can confirm that the uncut fragment is four kilobases because it lines up with the four-kilobase fragment in the DNA ladder sample.
We can also determine that the restriction enzyme has cut this fragment into three- and one-kilobase fragments by comparing the bands in the digested sample lane to those in the DNA ladder lane. For bands that do not perfectly align with one of the bands in the DNA ladder, size can either be estimated with the naked eye or determined using a mathematical calculation. In this way, scientists can use gel electrophoresis to verify or discover the size of DNA fragments.
In summary, agarose gel electrophoresis is a method of separating biological molecules using an electrical current. Ethidium bromide is a molecule commonly used to visualize DNA in agarose gel electrophoresis experiments. It intercalates between the nitrogenous bases of DNA and fluoresces under UV light.
Loading buffer is a solution added to an electrophoresis sample to give it color and density. A DNA ladder is a solution composed of DNA molecules of known length that is used to determine the size of DNA fragments in experimental samples.
After watching this lesson, you should be able to:
- Define agarose gel electrophoresis, DNA ladder and loading buffer
- Recall the attributes of ethidium bromide
- Analyze sizes of DNA samples in electrophoresis experiments