Lesson Video: Using Restriction Enzymes Biology • Third Year of Secondary School

Video Transcript

In this video, we’ll learn about restriction enzymes, a group of specialized enzymes involved in cutting DNA. We’ll also learn about how they target DNA, the specific sequences they recognize, and exactly what a palindrome is. After cutting DNA, restriction enzymes can leave behind so-called sticky ends, and we’ll see what makes these so important. So, let’s cut to the chase and start this lesson.

Restriction enzymes, also called restriction endonucleases, are a special group of proteins known as enzymes. An enzyme is a protein that acts as a biological catalyst, meaning it’s able to speed up a chemical reaction. Each enzyme has a specific substrate that it interacts with. The substrate is able to bind very specifically to the enzyme. And when it does, the enzyme is able to act on the substrate to speed up some chemical reaction, like the cleavage of the substrate, as shown here. These products can then leave the enzyme, and the enzyme is now free to accept another substrate.

A restriction enzyme is a special type of enzyme that catalyzes the cleavage of DNA, which is its substrate at specific recognition sequences in the DNA. In a way, they’re kind of like molecular scissors that can cut DNA at specific sequences. This can produce fragments of different lengths, as shown here. So, how is a restriction enzyme cutting DNA anyway? Let’s look a little bit closer at this section here and review the structure of DNA.

DNA is double-stranded. But for simplicity, we’ll just look at this single strand. DNA is made up of repeating subunits called nucleotides. This includes a phosphate group, a deoxyribose sugar, and a nitrogenous base. In DNA, there’s four bases: adenine, guanine, cytosine, and thymine. Shown here in black is what’s known as the sugar–phosphate backbone. This is where nucleotides are joined by phosphodiester bonds. Restriction enzymes can cleave this phosphodiester bond. This leaves a gap in the sugar–phosphate backbone, which we can see here. The restriction enzyme can also cleave the phosphodiester bond on the other strand, as shown here, and that’s how a restriction enzyme can cut DNA.

You might be wondering, what’s the role of restriction enzymes? Why do they even exist? They were discovered in bacteria in the 1950s, where research had shown that some bacteria were more resistant to viral infection compared to others. A bacteriophage is a type of virus that infects bacteria. Inside of its head, it contains viral DNA. This bacteriophage can attach to the surface of a bacterium and inject its DNA into the cytoplasm. Once the viral DNA has been injected, it can use bacterial resources to build new viral components. These can then be assembled to build new bacteriophages.

This process repeats, and the bacterium becomes filled with viruses until eventually the cell bursts and releases all the viruses to infect other cells. These bacteria are vulnerable to viral infection. However, some bacteria were found to be resistant to bacteriophage infection. These bacteria had restriction enzymes, and this would cut the viral DNA into fragments. These fragments could not be used to build new viral components, so the virus is effectively unable to infect the cell.

So, restriction enzymes are basically a mechanism developed by bacteria to defend themselves against viral infection. Note that bacteria can protect their own DNA from being cut by adding special methyl groups to their DNA. This can prevent the restriction enzymes from recognizing these sequences. Now, let’s look at how restriction enzymes combine to their recognition sequences.

A recognition sequence is a sequence of DNA that a restriction enzyme binds to and cleaves. These are also sometimes called restriction sites. Here’s a few examples of restriction enzymes and their recognition sequences. Each restriction enzyme recognizes a specific sequence and cuts the sequence between specific bases. So, for Eco RI, the sequence recognized G/AATTC, and it makes a cut between the guanine and the adenine, as shown here. There’s something special about these recognition sites that we’ll discuss next. But first, let’s review directionality in DNA.

Here’s a segment of a DNA molecule. You’ll notice that there are two strands. One of these strands goes in this direction, while the opposing strand goes in this direction. Let’s zoom in on this segment of the molecule so we can see it in more detail. The strand on the left is going down, and the strand on the right is going up. This directionality has to deal with the way nucleotides are added during DNA synthesis.

Nucleotides are only added in the five prime to three prime direction, which refers to the carbon numbers in the nucleotide. These numbers are indicated here. Notice that the five prime is above the three prime. So, this strand is sometimes referred to as the five prime to three prime strand. You can see that here. Whereas on the other strand, the five prime to three prime are in the opposite direction, which you can see over here.

Now, let’s look at the recognition sequence for Eco RI on both strands. Here’s the recognition sequence GAATTC, which by convention is written in the five prime to three prime direction. And here’s the sequence on the opposing three prime to five prime strand. Do you notice anything similar about these sequences? If you read the sequence on the top strand, GAATTC, it’s actually the same as the sequence on the bottom strand when read in reverse, GAATTC. This is actually an example of a palindrome, a word or phrase that reads the same forwards and backwards. An example of this is the word racecar, which still reads racecar whether you read it forwards or backwards. A palindrome sequence is actually a feature of many recognition sequences.

Let’s look at another example with the restriction enzyme Bam HI. In the top strand for Bam HI, it reads GGATCC, which is the same as the bottom strand when it’s read in reverse, GGATCC. So, this sequence is also a palindrome. Now, let’s look at the different cleavage patterns that can be made by restriction enzymes.

The first we’ll look at are the so-called sticky-end cuts. The recognition sequence and cut site for Eco RI is shown here. Let’s write this out for both strands so we can see how it’s cutting. Most restriction enzymes make two cuts one on each strand, which can be shown like this. This will generate two fragments. One is shown here, and the other is shown here. Notice that these two fragments have overhangs of unpaired bases. These can be called sticky ends because these bases are complementary and have an affinity for each other.

The second kind of cleavage pattern that can be made by restriction enzymes are called blunt ends. SmaI is an example of a restriction enzyme that makes a blunt-end cut. SmaI cuts to give one fragment that looks like this and another fragment that looks like this. These are called blunt ends, and because they don’t have any unpaired bases, they aren’t sticky. Now, let’s look at sticky ends in a bit more detail and talk about what makes them so important.

The phenomenon of sticky ends has a very interesting implication. Suppose we had two pieces of DNA that we wanted to stick together. But let’s make this fun. Let’s say we have a pet fish and we want to make them glow in the dark. Well, we know of a certain jellyfish that glows in the dark. This is caused by a protein called GFP, or green fluorescent protein. So, is it possible to isolate the GFP gene from the jellyfish DNA and insert it into the fish DNA? Using sticky ends, of course it is!

Lucky for us, there’s two Eco RI recognition sequences that border the GFP gene in the jellyfish DNA. And there’s also an Eco RI recognition site in the fish DNA. So, what we need to do is cut out the GFP gene with the Eco RI and insert it into the Eco RI site in the fish DNA. Let’s take a closer look and see how this works. Here’s the GFP gene with the two Eco RI recognition sequences at both ends. And here’s a closer look at the fish DNA with its single Eco RI site.

So now, we add Eco RI to the jellyfish DNA, and it cuts like this, and it produces this fragment with the isolated GFP gene. And we also cut the fish DNA with Eco RI, and it cuts like this. Now we add our cut GFP DNA to the cut fish DNA. And because these two ends are complementary, we can insert the GFP gene into the fish DNA. This new GFP gene gives our fish friend a whole new look.

Now, let’s apply what we’ve learned and look at a practice question.

Samples of DNA are taken from two organisms and mixed with restriction enzyme Bam HI. The restriction enzyme cuts the DNA from organism A into three sections but cuts the DNA from organism B into only two. What does this suggest about the DNA of the organisms? Organism A has fewer Bam HI recognition sequences in their DNA than organism B. Organism A has more Bam HI recognition sequences in their DNA than organism B. The DNA of organism B is longer than that of organism A. Or the sample taken from organism B was not mixed with enough restriction enzymes.

This question is asking us about restriction enzymes and how they can cut DNA differently between two organisms. There’s a few key terms in this question that we’ll need to address. So, let’s clear the answer choices so we have more room to work with. A restriction enzyme is a special type of enzyme. You’ll recall that an enzyme is a protein that acts as a biological catalyst. What this means is it can make a chemical reaction go much more quickly. It can do this by binding to its specific substrate and then create conditions for a chemical reaction to occur much more rapidly, like the cleavage of the substrate into two fragments as shown here.

Restriction enzymes are enzymes that catalyze the cleavage of DNA. Restriction enzymes are kind of like molecular scissors, and they can cut DNA, their substrate, at specific DNA sequences that they recognize. These specific DNA sequences are called recognition sequences, and each restriction enzyme has a unique recognition sequence that it binds to and cleaves.

The restriction enzyme Bam HI in this question has a recognition sequence of GGATCC. So, if the restriction enzyme that cut this DNA was Bam HI, then these two cuts would correspond to this sequence. In other words, this DNA has two Bam HI recognition sequences. After the DNA is cut, it will produce fragments or sections of DNA. So, in this example where there are two BAM HI recognition sequences in this linear DNA substrate, there are three sections that are formed.

Now, let’s look at the question again. The question states that Bam HI cuts the DNA from organism A into three sections but cuts the DNA from organism B into only two. Now, let’s assume the DNA is linear and work this out. Organism A is cut with Bam HI and produces three fragments. This implies that there must have been two recognition sequences for Bam HI, whereas in organism B, there’s only two fragments that are produced. This implies that there was only one Bam HI recognition sequence. So, when the DNA is linear, this suggests that there are more recognition sequences in organism A compared to organism B.

But what if the DNA is circular? Bacteria generally have circular DNA. So, let’s look at this situation before choosing an answer. Here is organism A being cut into three sections. In order to produce this fragment, the circular DNA would have to be cut here and here. And in order to produce the remaining two fragments, we would need an additional cut here. So, that’s a total of three recognition sequences for Bam HI in organism A. Here’s the two fragments produced in organism B. And in order to produce these fragments, we would need two cuts, one here and one here. So, organism B only has two Bam HI recognition sequences.

So, when the DNA is circular, organism A has more recognition sequences than organism B. This is the same result that we found with linear DNA. Therefore, the correct answer is that organism A has more Bam HI recognition sequences in their DNA than organism B.

Now, let’s look at some of the key points that we covered in this video. Restriction enzymes are enzymes that catalyze the cleavage of DNA. They do this by binding to specific DNA sequences called recognition sequences, meaning the sequence when read on one strand is the same when read on the opposing strand in the opposite direction. Restriction enzymes can cut DNA to produce two kinds of ends, blunt ends or sticky ends. Sticky ends can be used to join DNA from two sources and have many applications in biotechnology.