DNA Cloning - Online Tutor, Practice Problems & Exam Prep

DNA cloning involves isolating a specific segment of DNA, known as target DNA, from an organism. This process begins with obtaining the DNA, followed by cutting it with restriction endonucleases at specific restriction sites. The target DNA is then inserted into a plasmid vector, which replicates independently within a host organism, typically E. coli. DNA ligase is used to seal the DNA fragments together, creating a recombinant plasmid. This method is crucial for gene therapy, studying genes, and producing large quantities of specific DNA sequences for research and medical applications.

DNA Cloning

Hello, everyone. In this lesson, we are going to be talking about DNA cloning. Okay. So, some of you in the future, if you do biology lab or some sort of experiment, you may want to do DNA cloning. And I don't mean we're cloning an entire organism. I don't mean we're making a new Dolly or something like that. I'm talking about cloning small segments of target DNA. Target DNA is basically any gene or segment of DNA that you are interested in and you want to study. So the first thing to do whenever you're trying to clone a segment of DNA is you want to obtain that particular piece of DNA. So that's the first thing you want to do. And, generally, the way you're going to do that is you're going to take the organism, you're going to take a couple of cells from the organism of interest, and then you're going to cut that segment of DNA out. You might create multiple copies of that segment of DNA via the polymerase chain reaction for those specific sequences. And then once you have your sequence and you want to clone it, the next thing you're going to do is you're going to digest your segment of DNA with restriction enzymes. And this is going to cut the DNA into the very unique sequences that you want to clone. A restriction endonuclease is going to be a very important type of protein that basically cuts the DNA, and it's going to cut a segment of DNA out. Now, they are going to cut segments of DNA out, but they're not going to do it randomly. They're going to cut at the restriction sites. So, there are many different types of restriction endonucleases or restriction enzymes. And each one of them has their own unique restriction site that they recognize. So it may be a particular sequence, maybe it's A-G-G-C-T-A, and whenever that particular restriction enzyme sees that sequence in the DNA, it's going to cut it right there. So you may look at your piece of DNA, and you think I want this 10 base pair segment. How do I cut it out of there? Well, what are the sequences where I want it to cut? Once you figure out the sequence where you want it to cut, you find a restriction enzyme that cuts at that sequence. So it's a very specific protein, and it's a very useful type of protein in DNA work, in genetic work. It can cut DNA. It can also cut RNA. They're very unique restriction enzymes. So what you're going to do is you're going to get your sequence of DNA, and then you're going to cut your desired gene with restriction enzymes out of that segment of DNA. And then the next thing that you are going to do is you're going to paste it into the desired vector. This is basically a cut and paste scenario. You cut out the sequence that you want, and you're going to paste it into a new sequence. So, pasting isn't as easy as it may sound. You're going to paste your unique piece of DNA that you just cut out with a restriction enzyme into something called a vector, which is also commonly known as a plasmid. You're generally going to find these in prokaryotes. Plasmids are going to be small rings of DNA that are found inside of prokaryotic organisms that are not the chromosome of that prokaryotic organism. So basically, it's like a tiny segment of DNA that is not the chromosome, but is still replicated with that prokaryote. So what you're going to do is you're going to take your segment of DNA that you just cut out and you are going to paste it into this plasmid or this vector. And it's important to know that plasmids replicate independently of their organism and of the chromosome of that organism. Now, whenever you cut open a plasmid to place your segment of DNA into that plasmid, you're going to want to cut the plasmid with the same restriction enzyme that you cut the DNA with. To ensure that the ends of the DNA and the ends of the plasmid are able to with the same enzyme. And this is going to ensure that the ends of the plasmid and the ends of your segment of DNA of interest are able to bind with one another. Now, once you are able to actually get those 2 pieces of DNA in place, you're going to bind them together or glue them together with DNA ligase. Remember, we learned about DNA ligase whenever we were learning about DNA replication that happens naturally in cells. And DNA ligase's main job is to glue segments of double stranded DNA back together. We're going to use it for the same purpose here, but instead of gluing different segments of DNA back together, we're going to glue our segment of DNA into the vector or into the plasmid. So it's going to seal those 2 DNA fragments together. And now, we have something called a recombinant plasmid. It's going to be a plasmid that has the original DNA from the prokaryote, plus the sequence of DNA that we want to clone. So now what we're going to do is we are going to place the vector containing the DNA sequence into an organism, and this is generally going to be E. Coli. E. Coli is very, very commonly used for cloning. It does a really good job. So every time the E. Coli replicates, the plasmids are going to replicate, and you're going to clone a whole bunch of those plasmids or those vectors, and you're going to clone your segment of DNA many, many times. So, it's going to allow for incredibly large quantities of cloning of this specific DNA sequence. This is the way that we clone DNA into the largest number of clones that we are able to do. You may be thinking, why would we want to do this? Well, if the sequence of DNA is incredibly important, maybe it's a gene for a disease, and many scientists want to work on that gene, they each get a certain number of copies of the clone of that gene. So you're going to need to replicate it or clone that gene a lot. This cloning method is also utilized when you're studying the gene or when you want to do a transgenic experiment or create a transgenic organism, these kinds of things. Also, this is important for gene therapy. You have to clone the gene a lot for gene therapy. So basically, this is just utilized to better understand this particular segment of DNA and to use it in experiments. Okay? Alright. So, now, let's have a look at our figure. Okay. I'm going to go out of the figure because obviously I'm in the way, but I'll come back at the end. Okay. So this is just going to be a diagram of exactly what I already talked about. So, we have this sequence of original DNA from the original organism. So this is our organism of interest. Maybe it's a human being, maybe it's some other organism, but generally it's going to be probably a human gene that we want to clone so we can better understand that particular gene. And then we're going to have our restriction enzyme right here, and our restriction enzyme is going to locate its restriction and it's going to cut at those restriction sites. So, it's going to cut right here, and it's going to cut right here. And that is going to create this segment of DNA, which is no longer bound to the other sequences of DNA. So this is generally called our target sequence. This is going to be the sequence of DNA that we are interested in and that we particularly want to clone. Now, remember, I told you that the restriction enzyme also needs to cut the vector wherever it wants to insert that particular sequence of DNA. So the vector is going to be cut with the same restriction enzyme as the original organism's DNA was cut. So then, we're going to insert that segment of DNA into the vector where this restriction enzyme has cut, and then ligase is going to seal it up. So, let's rate that ligase glues Oh. Glues target sequence to the vector. Glues target to the vector. And, you guys can see here that it is all glued together, and that it is one plasmid or one vector. So this is commonly called a vector. And, you guys can see that they've placed this particular vector into the E. Coli Bacteria. Now, this vector is going to be independent of the chromosome of the E. Coli. So let's say that this is the chromosome of the E. Coli. The vector is not a component of DNA of the chromosome. It is going to be an independently replicating segment of DNA that is not connected to the chromosome. And then that vector and the E. Coli are going to replicate themselves, and you'll eventually have all of these different E. Coli with all of these vectors with your DNA segment in them. So, you're going to have a ton of vectors with this DNA segment. So you're going to have a ton of replicated or cloned segments of your target sequence. And then, basically, all you do is you remove the vectors from the E. Coli and you're going to actually cut your target sequence out of the vector, and then you're going to have gosh knows how many, probably a ton of your target sequence. So this is going to be the basic way that we clone massive amounts of DNA at the moment in time. This is going to be the upcoming lab. I hope that this was helpful. Let's now go on to our next lesson.

Which of the following shows the correct order of DNA cloning steps?

• A. DNA extraction → Digestion → Place DNA into E. coli → DNA ligase
• B. DNA ligase → Digestion → DNA extraction → Place DNA into E. coli
• C. DNA extraction → Digestion → DNA ligase → Place DNA into E. coli
• D. Digestion → DNA extraction → DNA ligase → Place DNA into E. coli

Here’s what students ask on this topic:

What is DNA cloning and why is it important?

DNA cloning is the process of creating multiple copies of a specific segment of DNA. This involves isolating the target DNA, cutting it with restriction enzymes, and inserting it into a plasmid vector. The recombinant plasmid is then introduced into a host organism, typically E. coli, where it replicates independently. DNA cloning is crucial for various applications, including gene therapy, studying gene functions, and producing large quantities of specific DNA sequences for research and medical purposes. It allows scientists to analyze genes in detail, develop treatments for genetic disorders, and create genetically modified organisms for research.

How do restriction enzymes work in DNA cloning?

Restriction enzymes, also known as restriction endonucleases, are proteins that cut DNA at specific sequences known as restriction sites. Each restriction enzyme recognizes a unique sequence, such as GGCTA, and cuts the DNA at or near this site. In DNA cloning, restriction enzymes are used to cut both the target DNA and the plasmid vector at specific sites, creating compatible ends. These ends can then be joined together using DNA ligase, forming a recombinant plasmid. This precise cutting and pasting mechanism is essential for inserting the desired DNA segment into the vector for cloning.

What role does DNA ligase play in DNA cloning?

DNA ligase is an enzyme that plays a crucial role in DNA cloning by sealing the nicks between DNA fragments. After the target DNA and plasmid vector are cut with the same restriction enzyme, they have compatible ends that need to be joined. DNA ligase facilitates this process by forming phosphodiester bonds between the adjacent nucleotides, effectively 'gluing' the DNA fragments together. This creates a stable recombinant plasmid that can be introduced into a host organism for replication. DNA ligase ensures the integrity and continuity of the DNA molecule, making it essential for successful cloning.

Why is E. coli commonly used in DNA cloning?

E. coli is commonly used in DNA cloning due to its well-understood genetics, rapid growth rate, and ability to take up and replicate plasmid DNA efficiently. E. coli cells can be easily transformed with recombinant plasmids, allowing for the production of large quantities of the cloned DNA. Additionally, E. coli has a relatively simple genome, making it easier to manipulate and study. Its widespread use in laboratories and the availability of various E. coli strains optimized for cloning further contribute to its popularity as a host organism in DNA cloning experiments.

What are plasmids and how are they used in DNA cloning?

Plasmids are small, circular DNA molecules found in prokaryotes, such as bacteria, that replicate independently of the chromosomal DNA. In DNA cloning, plasmids serve as vectors to carry the target DNA segment. After the target DNA is cut with restriction enzymes, it is inserted into the plasmid, which has been cut with the same enzyme to create compatible ends. DNA ligase then seals the DNA fragments, forming a recombinant plasmid. This plasmid is introduced into a host organism, like E. coli, where it replicates, producing multiple copies of the target DNA. Plasmids are essential tools for gene cloning, gene expression studies, and genetic engineering.