Agarose gel electrophoresis plays a key role in genetic engineering experiments. In this lesson, you’ll learn what agarose is and how electrophoresis works. You’ll also discover the use of agarose in this procedure.
Review of Genetic Engineering
So far, we’ve learned that genetic engineering involves changing the genetic makeup of an organism. We’ve been exploring the ins and outs of genetic engineering by imagining ourselves in the shoes of the scientists that engineered human insulin production in bacteria.
Let’s review what we’ve done up to this point to achieve this goal. First, we excised the human insulin gene from the human genome using restriction enzymes. After excising the insulin gene, we used DNA ligase to glue the gene into a plasmid. The plasmid will serve as the vector for transporting the gene into a host bacterial cell. But, we’re getting ahead of ourselves now.
It’s all fine and dandy to say that we’re using restriction enzymes and DNA ligase in our experiments, but how do we know if they’ve done their job or not? I mean, how do we know if the DNA got cut in the first step or if the gene and plasmid ligated in the second step? Wouldn’t it be terrible to get all the way to the end of our project and then find out we made a mistake in the first or the second step? Is there anything we can do? The answer is gel electrophoresis.
Mechanism of Electrophoresis
Gel electrophoresis is a laboratory procedure that uses an electrical current to separate biological molecules, typically nucleic acids or proteins, based on size.
You can remember electrophoresis’ mode of action based on the roots of the word. Not surprisingly, electro- refers to electricity, and -phoresis is derived from a Greek word that means ‘to carry.’ So, the name, essentially, means ‘carried by electricity.’
How is electricity going to help us? Recall the structure of DNA. A single DNA nucleotide is composed of a sugar, a phosphate and a nitrogenous base. Also, recall that a charged particle will move when placed in an electrical field. Therefore, if a DNA molecule possesses a charge, it’s going to move if we apply an electrical current to it.
The question is whether it has a charge and, if so, if the charge is positive or negative. Remember that phosphorus and oxygen atoms make up the phosphate group of a DNA nucleotide. Both elements are located on the right-hand side of the periodic table and, as such, are electronegative atoms that strongly attract electrons. This gives the phosphate group in a DNA nucleotide a net negative charge. Thus, DNA molecules are negative in charge.
How will a negative charge behave in an electrical field? Well, an electrical circuit consists of two electrodes: one positive and one negative. Negatively charged particles are attracted to positive charges and are repelled by negative charges. Therefore, DNA molecules will move toward the positive electrode of the circuit during gel electrophoresis.
Fantastic! Now we have a means of moving DNA fragments. But, we don’t have a means of distinguishing between different fragment sizes. We can move the DNA fragments but still can’t tell the difference between large ones and small ones. Fortunately, scientists discovered they could isolate a molecule from seaweed, called agarose. Agarose is a polysaccharide that can be used to form a gel to separate molecules based on size.
Basically, agarose behaves like gelatin. Scientists can heat and cool a mixture of agarose to form a gel. This allows them to form the agarose solution into a useful shape once it cools. Agarose is typically formed into a thin, flat brick with rectangular holes at one end. These holes, called wells, will provide a place to put the DNA samples at the beginning of the electrophoresis procedure.
How Agarose Separates DNA Fragments
So, once the agarose solution cools, it’s turned into a gel of interlinked agarose molecules. This gel consists of microscopic pores through which DNA can move. The rate at which DNA fragments can slip through the pores in this gel is based on size.
Let’s think about this idea another way. Imagine I have a colander with holes about the size of spaghetti noodles. I have spaghetti noodles of all different sizes, but I would like to separate the different-sized noodles. If I compare the rate at which an inch-long noodle and a foot-long noodle move through the colander, what do you expect to see? The shorter noodle should squirt through a hole faster than the longer noodle, right?
This same principle governs agarose gel electrophoresis. Small DNA fragments wiggle through the pores in the agarose gel faster than longer fragments. Fragments of the same size maneuver through the gel at the same rate. In this way, bands of DNA are produced that represent different-sized DNA fragments.
Why does the DNA appear as a band rather than say a circular or triangular shape? DNA simply moved straight through the gel from the starting wells. This produces a band the same width as the starting wells.
Gel Electrophoresis as a Checkpoint
How can gel electrophoresis be used to check the effectiveness of the restriction digest and ligation steps of our genetic engineering experiment? Agarose gel electrophoresis can be used to compare the starting sample and product of each step.
For instance, compare uncut DNA in the first lane to cut DNA in the second lane. Note that the larger fragment found in the first lane has been replaced by two smaller fragments in the second lane.
Next, consider the un-ligated DNA in the first and second lanes to the ligated product in the third lane. Note that we can tell that the ligation step was successful because the smaller starting fragments have been replaced by a single larger fragment in the product lane.
Using agarose gel electrophoresis to check the success of restriction digest and ligation steps is a staple in laboratory experiments. However, it is important to note that we are considering a greatly simplified explanation of genetic engineering in this lesson. In a genome as big as a human, any restriction enzyme would cut at sites besides the ones adjacent to the gene of interest. This would make other experiments necessary to identify the correct fragment, or the gene could be removed in a more specific fashion.
In summary, gel electrophoresis is a laboratory procedure used to separate biological molecules with an electrical current. Because of the negative charge on the phosphate group of DNA nucleotides, DNA molecules will move toward the positive electrode of the circuit during gel electrophoresis. Among other things, this procedure is commonly used by scientists to distinguish between DNA molecules of different sizes.
Agarose is a polysaccharide that can be used to form a gel to separate molecules based on size. The rate at which DNA fragments can slip through the pores in this gel is based on size. Small DNA fragments wiggle through the pores in the agarose gel faster than longer fragments.
At the conclusion of this video, you’ll be able to:
- Explain the process and importance of gel electrophoresis
- Describe the structure and function of agarose in gel electrophoresis