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 for ligation. DNA ligase then covalently joins these fragments, forming a recombinant DNA molecule. The second step, transformation, allows bacteria to uptake this recombinant DNA, resulting in transgenic organisms that can express foreign genes, such as those for insulin, demonstrating significant applications in medicine.

Steps to DNA Cloning

This video, we're going to talk about the steps to DNA cloning. Recall there are two general steps to DNA cloning, which we have outlined 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 transforming the DNA molecule, which means allowing bacteria to uptake the recombinant DNA. If we take a look at our image below, we can get a better understanding of these two steps involved with DNA cloning.

Over here on the far left, we have the first step to DNA cloning, which is creating the recombinant DNA, and recall that recombinant DNA is a single molecule that contains DNA from two different sources. In this image, our bacterial plasmid is shown in green, and our gene of interest, which would be from a different species like, for example, a human, is depicted here. This gene of interest is a gene coding for some protein that we're just calling protein X here.

The first thing that we need to do to create the recombinant DNA molecule is cut the DNA. You can see these little scissors here, used as a symbolic representation to show the cutting that must occur. In reality, it doesn't actually use scissors; it uses enzymes called restriction enzymes, which act like little tiny molecular scissors. We'll talk more about the restriction enzymes later as we move forward in our course.

After the DNA has been cut, as you see here, the second part, part 1b, is to ligate the DNA. Ligation of the DNA is basically pasting the DNA together, similiar to a cut-and-paste kind of thing. Part 1a is cutting the DNA, and part 1b is ligating the DNA or pasting the DNA together. We've got these little tiny glue bottles here, used as a symbolic representation of DNA ligases, which are the enzymes that ligate, paste together, or seal these two DNA molecules into a single molecule. The bacterial plasmid is over here, and the gene of interest coding for protein X is within the same molecule. This recombinant DNA molecule can then be used as a cloning vector to get the gene of interest into the host cell.

What we're showing in step number 2 is the transformation of the recombinant DNA, which involves allowing the bacterial cell, shown right here, to uptake the external DNA. It will uptake the external DNA and obtain the recombinant DNA molecule. Notice the scientist is expressing that now he has the recombinant DNA molecule within the bacterial host cell, he can grow this bacteria and allow the bacteria to express as much protein X as he needs. This also enables the creation of as much of the gene of interest, the product of the gene of interest, that we're trying to create.

We're going to continue to talk about these two steps, creating recombinant DNA and the transformation of recombinant DNA, moving forward. This introduction provides a foundation for practicing and discussing these concepts more in-depth in our next videos. 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. You can see that we're using these scissors in the image to represent the restriction enzymes, and the restriction enzymes are going to cut the DNA.

After the DNA has been cut, the second step, part 1b, is going to be to use ligase enzymes. Ligase enzymes are going to be enzymes that paste the DNA. Essentially, it's going to act like glue. Over here, we have a little bottle of glue which is supposed to be the symbolic representation of ligase enzymes or enzymes that are going to seal together and covalently link the cut DNA molecules. You can see here we've got the cut bacterial plasmid and the cut gene of interest that are being pasted together by the ligase enzymes, which are represented by these little glue bottles that you see here.

At this point, what we have is the final result, which is the recombinant DNA, and that concludes step number 1, the creation of recombinant DNA. This concludes this video and we'll be able to 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 details 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 specific locations called restriction sites on the DNA, and that is going to produce sticky ends. We are going to 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 do not just bind and cut to any region of the DNA. They only bind to very specific regions of the DNA and only cut very specific sequences, producing sticky ends. Sticky ends are really just a single-stranded DNA overhang that is produced from the restriction digestion reaction.

We can get a better feel for this down below in our image, looking at restriction enzymes, restriction sites, and sticky ends. Notice over here on the left-hand side, what we have is a specific DNA molecule. This DNA molecule has a specific region called the restriction site, and this specific region is going to have a specific DNA sequence that is going to be recognized by the restriction enzyme. The restriction enzyme is symbolized here in this image as a little pair of scissors even though it is a complex enzyme that is going to be binding to the restriction site and cutting it. When these restriction enzymes cut at the restriction site, they usually generate these sticky ends, creating a staggered type of cut in the DNA. Notice that the DNA is being cut in this kind of staggered way. When it's cut in this staggered way, it creates 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 they're called sticky ends is because they can still complementarily 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

In 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. It's important to note that DNA ligase is an enzyme that ligates or covalently joins the two sticky ends together that were created in step 1a. This creates the final recombinant DNA molecule that contains DNA from two different sources. 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. This is because the sticky ends generated by a restriction enzyme are quite unique, and only the correct sticky ends can be ligated together.

If we take a look at our example, we can see that a restriction enzyme and a DNA ligase are both needed and used to clone a recombinant DNA plasmid. On the left-hand side, we're showing you a bacterial plasmid, and on the right-hand side, we're showing you a eukaryotic cell, such as a human cell. There is a gene of interest highlighted in orange within the human cell, and this orange region represents the gene of interest. In step 1a, we know that restriction enzymes are going to be used, and these enzymes recognize restriction sites. Zooming into the plasmid DNA, you can see there is one restriction site. In the gene of interest, notice that it is flanked by two restriction sites.

The actual gene of interest is just a small region that you see in the middle. You use restriction enzymes to cut the plasmid DNA and another restriction enzyme to cut the gene of interest. As we know from step 1a from our previous videos, using restriction enzymes to cut these DNA molecules generates sticky ends, these single-stranded DNA overhangs. Notice that we have these sticky ends that are color-coded here, and what can happen is, these sticky ends can match and pair with each other where one sticky end comes and matches with this region, and another sticky end comes and matches with the other region. When this overlapping of these sticky ends across different molecules occurs, you can get what we have below, which is the gene of interest right in the middle, now being pieced back together with the DNA plasmid where it was cut.

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. The ligase enzymes are represented by little glue bottles because DNA ligase connects the DNA fragments just like glue is used to connect separate things together. Below, what we're showing you is really just the recombinant DNA molecule because it now has DNA from two 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 bacteria. The molecule ends up looking like what we see over here, where you have the bacterial plasmid and the gene of interest within it. We've created our recombinant DNA molecule.

Now that we've created this recombinant DNA molecule, we can talk about the transformation process, getting this recombinant DNA molecule into a bacterial host cell. But for now, this 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 as 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, 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. 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 that it has received the gene of interest with the antibiotic resistance.

And so these 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 so 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. Now diabetics actually do not produce enough insulin protein to metabolize blood glucose, and these diabetics require daily injections of insulin. Now researchers have found a way to use transgenic organisms in order to mass produce insulin for these diabetic patients that need these daily injections. And so, 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.

Now, of course, in the first step of DNA cloning, we know that we need to make the recombinant DNA. And so, we're going to take the bacterial plasmid here from the E. coli bacterium, and we're going to take the human gene, the insulin gene, and we're going to create a recombinant DNA molecule using restriction enzymes and DNA ligase. And then once the recombinant DNA has been made, it can be used as a cloning vector to be inserted into the bacteria. And so, the bacteria is going to be transformed with the recombinant plasmid. And so now, this bacteria contains a human insulin gene, making this organism a transgenic organism. And then, this bacterium with the transformed plasmid is just going to replicate normally via its normal replication process, creating a bunch of bacteria. And each of these bacteria is going to have a copy of the recombinant DNA. And because they have this gene, this human insulin gene, these bacteria are going to be reproducing and cloning the human insulin gene, and they'll be able to actually express the human insulin gene.

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

This here concludes our brief introduction to the application of DNA cloning in medicine and completes our review. And so, 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 includes using restriction enzymes to cut DNA at specific restriction sites, generating sticky ends. These sticky ends are then ligated by DNA ligase, which covalently joins the fragments to form recombinant DNA. The second step, transformation, involves introducing the recombinant DNA into bacteria, allowing them to uptake and express the foreign DNA. This results in transgenic organisms capable of producing proteins, such as insulin, for medical applications.

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 generates sticky ends, which are single-stranded overhangs that can base-pair with complementary sticky ends. DNA ligase then acts like glue, covalently joining these sticky ends to form a continuous DNA molecule. This process is crucial for creating recombinant DNA, which combines DNA from different sources, such as a bacterial plasmid and a gene of interest.

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 into bacterial cells. Once inside, the bacteria can replicate the recombinant DNA and express the foreign gene, producing the desired protein. This step is essential for creating transgenic organisms, which can be used for various applications, including the production of therapeutic proteins like insulin.

What are sticky ends and how are they generated?

Sticky ends are single-stranded overhangs of DNA that are 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 base-pair with complementary sticky ends from other DNA molecules, facilitating the ligation process by DNA ligase to form recombinant DNA. Sticky ends are crucial for the efficient and specific joining of DNA fragments in cloning.

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

DNA cloning is used to produce insulin by creating recombinant DNA that includes the human insulin gene. This recombinant DNA is introduced into bacteria, such as E. coli, through transformation. The bacteria then replicate and express the insulin gene, producing the insulin protein. This protein can be extracted and purified for medical use, providing a reliable and scalable source of insulin for diabetic patients who require daily injections to manage their blood glucose levels.