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.
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