In this video, we’ll discuss cloning of DNA sequences. First, we’ll define DNA cloning and recombinant DNA and then look at how plasmids can be used together with restriction enzymes and DNA ligase during the cloning process. Then, we’ll look at how bacterial cells can be transformed with recombinant DNA to make the clones. Finally, we’ll examine how reverse transcriptase and PCR can be used to isolate genes of interest for cloning.
The nucleated cells in our body contain DNA that give us the instructions needed for life. These instructions are organized as genes in DNA that control the expression of many of our traits. They can control the color of our eyes, our height, and of course our health and whether or not we’re affected by a disease. For example, the gene for insulin can produce a hormone that can control blood glucose levels.
Insulin is able to signal to the cell to take up glucose so it can be used for energy. A disease called diabetes can limit the amount of insulin in the body. And without sufficient insulin, the cell isn’t able to take up the glucose that it needs to carry out its functions. So, for diabetics, insulin needs to be given periodically to ensure that cells take up the glucose that they need. So where do we get this insulin from?
We can extract it from animals, like pigs, believe it or not. And this was common for many years because pig insulin is similar to human insulin. However, allergic reactions were still possible. So pig insulin was not ideal. We found our pig insulin replacement in the 1970s using something you probably wouldn’t expect, bacteria. So how is this possible? Well, we can take our gene for human insulin and insert it into bacterial DNA. The bacterium will then go on to produce human insulin. This can then be extracted and purified and used to treat diabetes.
Now is a good time to define a couple of key terms we’ll be using in this video. So let’s clear some room on the left of the screen. This process that we just described is referred to as DNA cloning. Here, the human insulin gene is inserted into bacterial DNA. And this is actually creating a genetically identical copy or a clone of the insulin gene. We can also use this as a verb and say that we cloned the insulin gene in this example.
Our second definition is recombinant DNA. When DNA cloning, we often use bacterial DNA to carry our gene of interest, or insulin in this example. So we have DNA from two sources: one from human DNA, as shown here in green, and one from bacterial DNA, shown here in black. These two sources of DNA are then combined together to form what we call recombinant DNA.
Now that we’ve covered DNA cloning in a general way, let’s talk more about the details of this process. We’ll start with discussing how recombinant DNA can be made. So here’s our recombinant DNA that’s been combined from two sources. We could see our gene of interest in green and the bacterial DNA in black. This bacterial DNA comes from a special type of DNA called plasmid DNA. If we look inside a bacterial cell, we’ll notice that there’s two types of DNA. There’s the bacterial chromosome, which is often a large circular piece of DNA that contains the information needed for the life cycle of the bacterium. And there’s plasmid DNA, which are also circular in structure and can act as accessory DNA molecules that can be shared between bacteria.
Plasmids may contain certain genes, such as antibiotic resistance genes, that can be beneficial for bacteria to grow in certain conditions and can also be used as a method that scientists use to select for bacteria that take up the plasmid. These plasmids are able to replicate independently of the chromosomal DNA. And a single bacterium can have hundreds of copies of a particular plasmid.
Plasmid DNA can also be called carrier or vector DNA because it can carry our gene of interest and can be taken up by our bacterium, where it’s treated like an ordinary plasmid. This means that it can express our gene of interest and also make multiple copies or clones of this gene as the plasmid replicates. In addition, these plasmids are passed on as bacteria divide, making even more copies.
Now, let’s talk about how plasmid DNA and our gene of interest can be joined to make recombinant DNA. So we’ll start with our plasmid DNA. But remember, DNA is double stranded. So let’s draw this to be a little bit more accurate. Okay, that looks better. And here we have our second source of DNA containing our gene of interest. So, in order to get our gene of interest inside the bacterial plasmid, we’re gonna need something to cut the DNA so we can insert this gene. And what cuts better than a pair of scissors? Molecular scissors, that is. You’ll recall that restriction enzymes or restriction endonucleases are special enzymes that act as molecular scissors to cut DNA. So our plasmid DNA can be cut here, and our gene of interest can be cut out from both sides.
Let’s see how this looks in the actual sequence of DNA. We’ll start with the plasmid DNA. Restriction enzymes cut DNA at their recognition sequence. The sequence GGATCC is the recognition sequence for the restriction enzyme BamHI. BamHI cuts DNA as indicated and produces these two ends. These are called sticky ends because they have unpaired nucleotides that can base-pair with complementary nucleotides on an opposing strand of DNA. So the sequence GATC is complementary to the sequence CTAG. This means that these two ends can hydrogen-bond with each other, which is what makes them sticky.
Now that we’ve cut our DNA on the right, let’s see what this looks like in the plasmid DNA diagram on the left. There, you’ll still notice that this diagram here is also showing the two sticky ends. So this section here corresponds to this sticky end, and this section here corresponds to this sticky end.
So now that we’ve opened up our plasmid using the BamHI restriction enzyme, let’s do the same thing for our gene of interest on the bottom. So now we can see the sequence on the right with our gene of interest being bordered by the two BamHI recognition sequences. And now we can cut these sequences with our restriction enzyme BamHI to give the indicated cutting patterns. This releases our gene of interest, which now has two sticky ends. These sticky ends are compatible with the two sticky ends in the cut plasmid DNA. So now we can combine them. That looks better.
Now, let’s do the same thing to the diagrams on the left to see how this looks. So the restriction enzyme cuts as indicated here, which can then be inserted into our cut plasmid DNA, as you can see here. So you may have noticed that there’s gaps in the diagram on the left, one of which is indicated by this pink square. We can also see this in the sequence on the right. These gaps represent missing phosphodiester bonds in the sugar phosphate backbone of DNA.
Let’s zoom in and see what this means. Here, we can see the chemical structure of the two strands of DNA. The strand on the left is the five prime to three prime strand. And on top, this corresponds to the top sequence, whereas the bottom sequence is the three prime to five prime strand, which corresponds to the structure on the right in the bottom diagram.
Nucleotides are joined together by phosphodiester bonds as indicated here. Restriction enzymes cleave this phosphodiester bond, and this is where this gap comes from. So, at this point, the only thing holding our gene of interest in the plasmid is the hydrogen bonding between the sticky ends. To combine these two DNA molecules permanently, we need to repair this missing phosphodiester bond. To do this, we can use an enzyme called DNA ligase, which can catalyze the formation of a phosphodiester bond. DNA ligase can therefore fill in all the gaps in the sugar phosphate backbone that were introduced by the restriction enzyme. You can see this here on the top and here on the left as well.
Now, our recombinant DNA is all stitched up and ready for the next step, where we transfer this into bacteria. This step is known as transformation. During transformation, the bacteria are exposed to a certain chemical and temperature that can help make them more permeable to DNA. This way, when the bacterium is mixed with the recombinant DNA, it is readily taken up into the cytoplasm.
Now, the recombinant DNA can be expressed using bacterial components to form the corresponding protein from our gene of interest. If the gene was insulin, for example, we’d now be able to harvest the insulin protein. A single recombinant DNA molecule is shown here for simplicity. But there will actually be multiple copies, since it will replicate just like a plasmid would. In addition, the bacteria will divide to make even more copies. Ultimately, this will produce a large amount of our protein.
Now that we’ve seen how DNA cloning takes place, the next topic is how to isolate our gene of interest in order to clone it. Normally, genetic information flows from DNA to mRNA to protein. A gene in DNA can be converted to mRNA by transcription. The reverse process can also take place, where mRNA is converted back into DNA. This process is called reverse transcription. So, if we want to clone a particular gene, one way to do this is to isolate the mRNA and reverse-transcribe it back into DNA.
It’s important to be working with DNA because restriction enzymes and plasmids, which we need for cloning, are usually exclusively DNA based. So how do we isolate this mRNA in order to make the DNA for our gene of interest?
Suppose our gene of interest is insulin. We know that the pancreas contains some cells that produce a lot of insulin mRNA. So this is a good place to start. In the lab, we could harvest these cells and isolate this mRNA. We can then perform reverse transcription on the mRNA to make insulin DNA that we can then insert into a plasmid for DNA cloning.
Now, let’s write up the sequence of mRNA so we can work out how reverse transcription actually takes place. So here is our mRNA, and here is the enzyme that performs reverse transcription that we call reverse transcriptase. Because it adds DNA nucleotides that are complementary to mRNA, we call its product cDNA or complementary DNA. As reverse transcriptase moves along the mRNA in the five prime to three prime direction, it adds these complementary nucleotides to the growing cDNA molecule.
Now that the cDNA molecule is complete, let’s take a second to go over a few things. Remember that mRNA contains uracil in place of thiamine as in DNA. And uracil base-pairs with adenine as shown here. In DNA, we have thiamine, which can base-pair with adenine in mRNA. A common mistake is to think that this adenine would pair with uracil, but reverse transcription produces cDNA and not mRNA. So we want to be sure that we’re using the right nucleotides.
Now, what we want to do is get rid of the mRNA in this mRNA and cDNA complex and replace it with another strand of DNA to make this whole molecule double-stranded DNA. That way, we can use it for DNA cloning. So, in the lab, we can use a special enzyme to specifically degrade this RNA molecule to remove it.
Now, with mRNA gone, we’re left with the single-stranded cDNA. The next step is to make this DNA double stranded. And for that, we can use our old friend DNA polymerase, who you might remember plays a big part in replicating DNA before cell division. So DNA polymerase can bind to the single-stranded cDNA molecule and add complementary nucleotides to make the second strand of DNA. And now we have our double-stranded DNA molecule that’s ready for DNA cloning.
We can also make multiple copies of a DNA molecule using another technique called the polymerase chain reaction, or PCR. This is a lab technique that allows us to target specific regions of an organism’s DNA to make multiple copies of it. By doing this, we’re actually able to make clones of our gene of interest without even using bacteria. However, in some situations, we might want to use these copies to form recombinant DNA, which can then be used to transform bacteria. This is useful because the protein can then be made. So, in this way, PCR is another way that we can isolate our gene of interest.
Now that we’ve seen how cloning works and how to isolate genes to be used in cloning, it’s time to look at a practice question.
Using plasmids to form recombinant DNA is a crucial part of cloning DNA. In which microorganisms were plasmids originally discovered?
Let’s look at an example of cloning the interferon gene in order to answer this question. Interferons are proteins that can interfere with viral replication. As such, they can be used effectively as antiviral drugs. They can also be used to treat certain diseases, such as cancer and multiple sclerosis. In the past, interferon was extracted from cells, which made it very expensive. These days, a process called DNA cloning can be used. This makes it possible to make a copy or clone of the gene for interferon. This process starts with isolating the gene for interferon and inserting it into a circular piece of bacterial DNA called a plasmid.
Plasmids were originally discovered in bacteria and are extrachromosomal pieces of DNA that replicate independently. A single cell can have hundreds of copies of a single plasmid. In cloning, they’re used to carry genes that we’re interested in making copies of. So, in this context, they’re sometimes called vectors or carrier DNA. On the left, you’ll notice that this construct we’ve made contains DNA from two sources. One is the DNA from the human interferon gene, and the other is the plasmid DNA from the bacteria. When two sources of DNA are combined like this, we call it recombinant DNA.
In the final step of the cloning process, this recombinant DNA can then be transferred into bacterial cells. Here, the bacterial cell can express this gene to produce the interferon protein. Only one copy of the recombinant DNA is shown here. But it will replicate in the cell to make many copies. And there will be even more copies as the bacteria divide. This will lead to a lot of interferon production, which can then be extracted and used for medical purposes. Plasmids are a crucial part of DNA cloning because they’re used to carry the gene we want to clone. Plasmids were originally discovered in bacteria.
Now, let’s go over some of the key points that we covered in this video. DNA cloning is the process of making copies or clones of specific DNA that we’re interested in. This DNA can be cloned into a plasmid. These two sources of DNA, the specific DNA we’re interested in and the plasmid DNA, can be combined to form recombinant DNA. Recombinant DNA can be made by cutting DNA using restriction enzymes and then inserting this in the plasmid DNA. DNA ligase can then be used to form new phosphodiester bonds in the cuts made by the restriction enzyme. Recombinant DNA can then be transformed into bacteria, where multiple copies can be made and expressed using bacterial components. Finally, the specific DNA that we’re interested in cloning can be isolated using reverse transcription or PCR.