Steps to DNA Cloning - Online Tutor, Practice Problems & Exam Prep

DNA cloning involves two main steps: creating recombinant DNA and transforming it into bacteria. The first step utilizes restriction enzymes to cut DNA at specific restriction sites, generating sticky ends that allow for ligation by DNA ligase, forming a recombinant DNA molecule. The second step, transformation, enables bacteria to uptake this recombinant DNA, resulting in transgenic organisms capable of expressing foreign genes, such as antibiotic resistance. This technique has significant applications in medicine, including the mass production of insulin for diabetic patients.

Steps to DNA Cloning

This video, we're going to talk about the steps to DNA cloning. Recall there are 2 general steps to DNA cloning, and we have them down below labeled number 1 and number 2. The first step in DNA cloning is making or creating the recombinant DNA molecule. The second step is going to be forming the DNA molecule, which just means allowing bacteria to uptake the recombinant DNA. If we take a look at our image down below, we can get a better understanding of these two steps involved with DNA cloning.

Over here on the far left, what we have is the first step to DNA cloning, which is creating the recombinant DNA. Recall that recombinant DNA is a single molecule that contains DNA from 2 different sources. Here in this image, what we have is our bacterial plasmid in green, and our gene of interest, which would be from a different species, like for example, a human. Here, this gene of interest is a gene coding for some protein that we're just calling protein x. What you can see is that, in order to create the recombinant DNA molecule, the first thing that we need to do is cut the DNA.

You can see these little scissors here, that we're using as a symbolic representation, to show you the cutting that must occur to create the recombinant DNA molecule. In reality, it doesn't actually use scissors. It uses these enzymes called restriction enzymes, which kind of act like little tiny molecular scissors, but we'll talk more about the restriction enzymes later as we move forward in our course. The first step is to cut the DNA. After the DNA has been cut as you see here, the second part here, part 1b, is to ligate the DNA. Ligate the DNA is basically like pasting the DNA together. So it's almost like a cut and paste kinda thing. One here is cutting the DNA and 1b is ligating the DNA or pasting the DNA together.

You can see we've got these little tiny glue bottles here just being used as a symbolic representation of DNA ligases, which are the enzymes that ligate or paste together or seal together these 2 DNA molecules. You've got these cut fragments over here that are going to be pasted together into a single molecule. You've got the bacterial plasmid over here and the gene of interest coding for protein x is within the same molecule. Then this recombinant DNA molecule can be used as a vector, a cloning vector, to get the gene of interest into the host cell.

And that's what we're showing you over here in step number 2 is the transformation of the recombinant DNA. Transformation here in this context is just talking about allowing the bacterial cell, which is right here, to uptake the external DNA. It's going to uptake the external DNA and be able to obtain that recombinant DNA molecule. Notice that the scientist way over here is now saying, now that he's got this recombinant DNA molecule within the bacterial host cell. He's saying he can grow this bacteria and allow the bacteria to express as much protein x as he needs.

Basically, through DNA cloning, we are able to clone the DNA and also create as much of the gene of interest, the product of the gene of interest, that we're trying to create. This is something that we're going to continue to talk about more and more, these two steps. Step number 1, creating recombinant DNA. We'll talk more about that moving forward. And we'll also talk more about transformation of recombinant DNA moving forward as well. This is just the introduction here. And that concludes this introduction to the steps to DNA cloning. Again, we'll be able to get practice applying these concepts and talk more about these concepts as we move forward. So I'll see you all in our next video.

Step 1) Create Recombinant DNA

In this video, we're going to talk more about the details of step number 1 of DNA cloning, which is creating the recombinant DNA. Creating a recombinant DNA molecule is actually a two-step process within itself that requires step 1a and step 1b.

Now, step 1a involves using restriction enzymes to cut the DNA. These restriction enzymes act like tiny molecular scissors because they can cut the DNA molecule. In the image, we use scissors to represent the restriction enzymes, showing how they cut the DNA. After the DNA has been cut, the second part, step 1b, involves using ligase enzymes. Ligase enzymes act like glue, covalently linking the cut DNA molecules. Here, we depict this with a little bottle of glue symbolizing the ligase enzymes, which paste together the cut bacterial plasmid and the gene of interest.

At this point, we have the final result, which is the recombinant DNA, concluding step number 1, the creation of recombinant DNA. This concludes this video, and we will move on to step number 2 in our next video.

Human DNA cut with restriction enzyme A can be joined to:

• A. A bacterial plasmid cut with restriction enzyme A.
• B. Human DNA cut with restriction enzyme B.
• C. Human DNA that is uncut.
• D. A bacterial plasmid that is uncut.

1a) Use Restriction Enzymes

In this video, we're going to talk more about step 1a in the steps of DNA cloning, which is using restriction enzymes. Recall that restriction enzymes are enzymes themselves that are specifically important for cleaving or cutting the DNA at these specific locations called restriction sites on the DNA, and that is going to produce sticky ends. We will define restriction sites and sticky ends down below in our text. Restriction sites are really just defined as specific sequences of DNA where a restriction enzyme will bind and cut the DNA. These restriction enzymes don't just bind and cut any region of the DNA. They only bind to very specific regions of the DNA and only cut very specific regions of the DNA called restriction sites. Of course, once that DNA has been cut at the restriction site, it produces sticky ends, and sticky ends are really just a single-stranded DNA overhang that is going to be produced from the restriction digestion reaction. We can get a better feel for this down below in our image, looking at restriction enzymes and restriction sites along with sticky ends.

Notice over here on the left-hand side, what we have is a specific DNA molecule here, and this DNA molecule has a very specific region called the restriction site, and this specific region is going to have a very specific DNA sequence that is going to be recognized by the restriction complex enzyme that is going to be binding to the restriction site and cutting the restriction site. When these restriction enzymes cut at the restriction site, they usually generate these sticky ends and so they create a staggered type of cut in the DNA. Notice that the DNA is being cut in this kind of staggered way, and when it's cut in this staggered way, it creates these overhangs, these single-stranded DNA overhangs that are kind of sticking out of the rest of the molecule. These are the sticky ends that we are referring to. The reason that they're called sticky ends is because they can still complementary base pair to other matching sticky ends as we'll talk about moving forward in our course. But for now, this here concludes our brief lesson on step 1a, using restriction enzymes, and how restriction enzymes will bind and cut restriction sites to generate sticky ends and separate molecules. We'll be able to get some practice applying these concepts and talk more about step 1b as we move forward. So, I'll see you in our next video.

What is the enzymatic function of restriction enzymes?

• A. To add new nucleotides to the growing strand of DNA.
• B. To join nucleotides during replication.
• C. To join sticky ends of DNA.
• D. To cleave DNA at specific sequences.

Each restriction enzyme has a specific sequence of nucleotides where it cuts the DNA. These sequences of DNA are unique to each restriction enzyme and are known as:

• A. Restriction Sites.
• B. Restriction Signals.
• C. Restriction Sequences.
• D. Restriction Sticky Ends.

1b) Use Ligation Enzymes

This video, we're going to talk more about step 1b of the steps of DNA cloning, which is using ligation enzymes to help finalize the creation of the recombinant DNA molecule. And so what's important to note is that DNA ligase is an enzyme, and it is an enzyme that ligates or covalently joins the 2 sticky ends together that were created in step 1a. And that is going to create the final recombinant DNA molecule that contains DNA from 2 different sources. Now, it is important to note that only DNA fragments that have been cut by the same restriction enzyme are capable of being ligated back together. And that's because the sticky ends that are generated by a restriction enzyme are going to be quite unique, and only the correct sticky ends can be ligated together.

And so if we take a look at our example down below, we can see that a restriction enzyme and a DNA ligase are both needed, and used to clone a recombinant DNA plasmid. And so down below here, we're looking at creating recombinant DNA. And what's important to note is that over here on the left-hand side, we're showing you a bacterial plasmid. And over here on the right-hand side, we're showing you a eukaryotic cell, such as, for example, a human cell. And say there is a gene of interest highlighted here in orange that is within the human cell, and, this orange region here represents the gene of interest. Now what's important to note is that, of course, in step 1a, we know that restriction enzymes are going to be used, and the restriction enzymes are going to recognize restriction sites. And so over here, zooming into the plasmid DNA, you can see that there's one restriction site as you can see. And over here in this gene of interest, notice that it is being flanked by 2 restriction sites. And, the actual gene of interest is just this small little region that you see right here in the middle.

And so you use restriction enzymes to cut the plasmid DNA and restriction enzyme to cut the to cut these DNA molecules is going to generate sticky ends, these single-stranded DNA overhangs. And so notice that, we have these sticky ends that are color-coded here in these colors. And what can happen is, these sticky ends, they can match and pair with each other where this sticky end comes and matches with this region, and this sticky end over here comes and matches with the other region. And so when that happens, this overlapping of these sticky ends, across different molecules, you can get what we have down below, which is the gene of interest right here in the middle, now, is being pieced back together with the right in-between the DNA plasmid where the DNA plasmid was cut. And so, of course, in order to covalently join and seal the gene of interest in the middle with the plasmid, what we'll need is these ligase enzymes. And the ligase enzymes are being represented by these little glue bottles because the DNA ligase is going to connect the DNA fragments just like glue is used to connect separate things together.

And so, down below here, what we're showing you is really just the recombinant DNA molecule because it now has DNA from 2 different sources. It has the gene of interest, which was isolated from the human cell, and of course, it has the plasmid DNA, which was from the bacteria. And so, the molecule ends up looking like what we see over here, where you have the bacterial plasmid and the gene of interest within it. And so we've created our recombinant DNA molecule. And so now that we've created this recombinant DNA molecule, we know just in general how this a bacterial host cell. But for now, this here concludes our introduction here to step 1b, using ligation enzymes, and we'll be able to get some practice applying these concepts as we move forward in our course.

The single-stranded ends of DNA molecules can be joined together by:

• A. Restriction Endonucleases.
• B. DNA ligase.
• C. DNA polymerase.
• D. Helicase.
• E. Primase.

2) Transform Recombinant DNA into Bacteria

So now that we've talked about the first step of DNA cloning, which is making the recombinant DNA molecule in our previous lesson videos, in this video, we're going to talk about the second step of DNA cloning, which is transforming the recombinant DNA into bacteria. And so again, the second and final step of DNA cloning is to transform the recombinant DNA. Now, this term transform is not used like you would use the term transform in your everyday language. In this context, the term transform is referring to the process of transformation. And transformation is the process that allows cells to directly uptake foreign DNA from their environment and allows them to obtain foreign DNA from their environment. For example, it allows them to obtain a cloning vector, such as a recombinant DNA molecule. And so, organisms that have successfully transformed a recombinant DNA molecule are called transgenic organisms. And so, a transgenic organism is an organism that has received and expressed the recombinant DNA molecule. And so the transgenic organism is going to have DNA that is going to come from a completely different source, a completely different species. Now scientists can use what are known as phenotypic markers, for example, antibiotic resistance in order to confirm a positive transformation and confirm that the bacteria have successfully received the recombinant DNA molecule. And so we can get a better understanding of the process of transformation down below in our image. And in this example image, we're looking at creating a transgenic organism with antibiotic resistance by transformation of recombinant plasmid DNA. And so in this, image down below, of course, we know that in the first step of DNA cloning, we need to make the recombinant DNA, and that's what we're showing you here in this first part of the image. And so you can see that we've got the bacterial plasmid DNA over here and the gene of interest over here, which in this case is going to have an antibiotic resistance gene. And so of course if we want to create the recombinant DNA molecule, then we're going to need to use restriction enzymes to cut each of these DNA molecules and then use DNA ligase to ligate or join them together to create the recombinant DNA molecule, which is going to have the gene of interest, connected to the bacterial plasmid. And so this recombinant DNA can serve as a cloning vector to get the gene of interest into the bacterial cell. And in this image, we're showing you an E. coli bacterium, and you can see the bacterial genome right here. And really, this whole video is focusing on this step right here, the second step of DNA cloning which is transformation. The process of this bacterium uptaking external DNA like this cloning vector here. And so through the process of transformation, notice that the cloning vector is going to get into the E. coli bacterium as we see down below right here. And so this E. coli is now an organism that has DNA from a completely different source, a completely different species. It has this gene of interest. And so now this organism is a transgenic organism because it has this recombinant DNA. And so this would be, the transformed E. coli and now it has antibiotic resistance now that it has received the gene of interest with the antibiotic resistance. And so these, this transformed bacterial cells, they can actually replicate and express the gene of interest, and the researcher can then purify the gene's product and study the gene of interest and the product of the gene of interest. And so, this here is really the conclusion to the second step of DNA cloning which is forming the recombinant DNA into the bacteria. And, we'll be able to get some practice applying these concepts that we've learned as we move forward in our course.

The process of using DNA from one organism to alter the characteristics of another is called:

• A. Translation.
• B. Transduction.
• C. Transcription.
• D. Transformation.

An organism which has foreign genes incorporated into its genomes is known as a:

• A. Recombinant organism.
• B. Transgenic organism.
• C. Hybrid organism.
• D. Polyploid organism.

Review & Application of DNA Cloning in Medicine

In this video, we're going to do a review and talk about the application of DNA cloning in medicine. And now that we've discussed the techniques used in DNA cloning, let's see how they're all used together in this application of DNA cloning in medicine. Diabetics do not produce enough insulin protein to metabolize blood glucose, and these diabetics require daily injections of insulin. Researchers have found a way to use transgenic organisms to mass produce insulin for these diabetic patients that need these daily injections.

Down below in our example image, we're going to talk about how human insulin protein is expressed and purified in large amounts using transgenic E. coli, transgenic bacteria. So, we're focusing on the cloning of the human insulin gene. Of course, in the first step of DNA cloning, we know that we need to make the recombinant DNA. We're going to take the bacterial plasmid from the E. coli bacterium, and the human gene, the insulin gene, and we're going to create a recombinant DNA molecule using restriction enzymes and DNA ligase. Once the recombinant DNA has been made, it can be used as a cloning vector to be inserted into the bacteria.

The bacteria are going to be transformed with the recombinant plasmid. Now, this bacteria contains a human insulin gene, making this organism a transgenic organism. This bacterium with the transformed plasmid will replicate via its normal replication process, creating a bunch of bacteria. Each of these bacteria will have a copy of the recombinant DNA. Because these bacteria have this gene, the human insulin gene, they are going to reproduce and clone the human insulin gene, and they'll be able to express the human insulin gene.

We have bacteria that have the human insulin gene. The cloned insulin genes can be used for other experiments. And the human insulin hormone—the protein that's being expressed and created by the bacteria—can also be extracted and purified, and that insulin can be given to diabetic individuals to help those patients. What we're seeing here is that cloning the human insulin gene does have medical applications.

This concludes our brief introduction to the application of DNA cloning in medicine and completes our review. We'll be able to get some practice applying these concepts as we move forward in our course. So, I'll see you all in our next video.

What is the most logical sequence of steps for splicing foreign DNA into a plasmid and inserting the plasmid into a bacterium?
I. Transform bacteria with a recombinant DNA molecule.
II. Cut the plasmid DNA using restriction enzymes (endonucleases).
III. Extract plasmid DNA from bacterial cells.
IV. Hydrogen-bond the plasmid DNA to non-plasmid DNA fragments. V. Use ligase to seal plasmid DNA to non-plasmid DNA.

• A. II, III, V, IV, I
• B. III, II, IV, V, I
• C. III, IV, V, I, II
• D. IV, V, I, II, III

Here’s what students ask on this topic:

What are the main steps involved in DNA cloning?

DNA cloning involves two main steps: creating recombinant DNA and transforming it into bacteria. The first step uses restriction enzymes to cut DNA at specific restriction sites, generating sticky ends. These sticky ends allow for the ligation by DNA ligase, forming a recombinant DNA molecule. The second step, transformation, enables bacteria to uptake this recombinant DNA, resulting in transgenic organisms capable of expressing foreign genes, such as antibiotic resistance. This technique is crucial for various applications, including the mass production of insulin for diabetic patients.

How do restriction enzymes and DNA ligase work in DNA cloning?

Restriction enzymes act like molecular scissors, cutting DNA at specific sequences known as restriction sites. This cutting produces sticky ends, which are single-stranded DNA overhangs. DNA ligase then acts like glue, covalently joining these sticky ends to form a continuous DNA strand. Only DNA fragments cut by the same restriction enzyme can be ligated together, ensuring that the sticky ends match. This process is essential for creating recombinant DNA molecules, which can then be introduced into bacterial cells for cloning.

What is the role of transformation in DNA cloning?

Transformation is the process by which bacteria uptake foreign DNA from their environment. In DNA cloning, this step involves introducing the recombinant DNA molecule into bacterial cells. Once inside, the bacteria can replicate the recombinant DNA, producing multiple copies. These transgenic bacteria can express the foreign gene, such as an antibiotic resistance gene or a gene coding for a protein like insulin. Transformation is crucial for producing large quantities of the gene product for research or medical applications.

What are sticky ends and how are they generated?

Sticky ends are single-stranded DNA overhangs created when restriction enzymes cut DNA at specific restriction sites. These enzymes make staggered cuts, leaving short, single-stranded regions at the ends of the DNA fragments. These sticky ends can form hydrogen bonds with complementary sticky ends on other DNA fragments, facilitating the ligation process by DNA ligase. This property is essential for creating recombinant DNA molecules, as it allows for the precise joining of DNA from different sources.

How is DNA cloning used to produce insulin for diabetic patients?

DNA cloning is used to produce insulin by creating a recombinant DNA molecule that includes the human insulin gene. This recombinant DNA is introduced into bacterial cells through transformation. The transgenic bacteria then replicate, producing multiple copies of the insulin gene. These bacteria express the insulin protein, which can be extracted and purified. The purified insulin is then used for daily injections in diabetic patients, providing a reliable and scalable source of this essential hormone.