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.