Gel electrophoresis is used to analyze DNA restriction digest and ligation experiments. In this lesson, you will learn how to use a DNA ladder to interpret experimental results.
Purpose of Gel Electrophoresis
Gel electrophoresis is a laboratory procedure used to separate biological molecules with an electrical current. Previously, we’ve discussed gel electrophoresis in the context of analyzing DNA. Using an electrical current and a molecule that behaves like gelatin, called agarose, we can separate DNA molecules based on size.
Although agarose gel electrophoresis can be used for a variety of purposes, two of the most common uses with respect to DNA are verifying the success of a restriction digest or a ligation step in an experiment.
Analyzing a Restriction Digest
Agarose gel electrophoresis is an effective means of determining if a restriction digest procedure has been successful. Let’s consider a simple example of how this works.Often, scientists want to express a protein in a cell or tissue where it’s not normally found. Suppose we want to make corn that is resistant to pest infestation, so we don’t have to use so many chemical pesticides.To do this, let’s combine the DNA from two different genes, so a pest toxin can be expressed in our crop. We could accomplish this by cutting each gene with the same restriction enzyme and combining the DNA fragments to create a brand new recombinant gene.
But, before any recombinant DNA can be created, we’d want to verify that the restriction digest was successfully completed.
Let’s say gene 1 is a corn gene that is 4 kilobases long, but we only want to use the first 0.
5 kilobase in our new recombinant gene. And, gene 2 is a pest-resistance gene that is 3 kilobases long, but we only want to use the last 2.5 kb in the recombinant gene. In both cases, the restriction digest should replace the band in the uncut lane with two smaller bands in the digested lane.
Using a DNA Ladder Standard
Besides just concluding that one piece of DNA has been cut into two smaller DNA fragments, we can also determine the size of both the starting sample DNA and the product of a restriction digest. Recall that we can use a set of DNA molecules of known length, called a DNA ladder, to determine the size of DNA fragments in an electrophoresis gel. A DNA ladder allows us to make more precise conclusions about our gel electrophoresis results.
Notice the DNA ladder in lane one of the example below. Since we know the size of each of these bands, we can use them as a reference point for the experimental samples.Consider lane two of the gel.
By comparing the band in lane two to the DNA standards of known size, we can conclude that our initial DNA sample was in fact the correct size. Furthermore, we can also conclude that the sample did not contain any detectable nucleic acid contaminants that could disrupt future experimental steps.Next, consider lane three of the gel.
The bands in lane three represent DNA from the corn gene that has been digested with a restriction enzyme. Rather than simply conclude that the starting DNA was cut, by using the DNA ladder standards as a point of comparison, we can determine if the DNA was cut in the correct location.
Note that the 4 kb band in lane two has turned into two bands of 3.5 kb and 0.
5 kb in lane three. Therefore, we can conclude that the restriction digest produced DNA fragments of the expected size. Additionally, it is important to note that we can also conclude that all of the starting DNA was cut because the band at 4 kb is no longer present in lane three.
A Troubleshooting Tool
We’ve discussed how gel electrophoresis can be used to verify that experiments have worked properly. Another major function of gel electrophoresis is to help identify the cause of a problem when something goes wrong.
Now, consider lanes four and five of our sample gel. These lanes represent the restriction digest performed on the pest-resistance gene. What size bands do we predict to see in each lane?Recall that our starting DNA is three kilobases this time. And, the restriction enzyme we used should produce a 2.5 kb and 0.5 kb fragment. Surprisingly, unlike our previous digest, lane five possesses three bands instead of two.
In addition to the 2.5 kb and 0.5 kb bands we predict the restriction digest to produce, we also see a faint band at 3 kb.
What does this mean? This 3 kb band most likely represents uncut DNA. Agarose gel electrophoresis has alerted us that the digest was incomplete and future experimental steps may be compromised if we don’t redo this digestion step.Because the starting DNA produced a 3 kb band of the expected size, we can conclude that our starting DNA sample appears to be fine. Furthermore, based on the results, we can instead concentrate on determining if anything was wrong with the restriction enzyme or if the digestion was set up incorrectly. Thus, gel electrophoresis both has identified the problem and has narrowed the troubleshooting investigation.
Analyzing a Ligation Reaction
Another primary reason to use gel electrophoresis is analysis of a ligation reaction.
If two pieces of DNA have been cut with the same restriction enzyme, they will have complementary sticky ends. Hydrogen bonds can form between complementary sticky ends, and the enzyme ligase can be used to join the molecules permanently by forming a bond between adjacent phosphates and sugars in the two DNA molecules.
But, how will we determine if the pieces of the corn and pest-resistance genes have been successfully ligated together? By comparing the starting DNA samples and ligation products on an agarose electrophoresis gel, we can determine the effectiveness of this step as well.
Again, the DNA ladder is located in lane one of this gel. Notice that a successful ligation experiment results in the formation of a 3 kb fragment, representing the new recombinant gene.
In summary, gel electrophoresis is a laboratory procedure used to separate biological molecules with an electrical current. Two major uses of gel electrophoresis are analyzing restriction digest and ligation experimental results.
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. It can be used to verify the size of DNA fragments prior to or following an experimental protocol, such as a restriction digest or DNA ligation.
At the end of this video, you’ll be able to analyze the results of restriction digest and ligation experiments using gel electrophoresis.