1b) Use Ligation Enzymes

by Jason Amores Sumpter
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in this video, we're going to talk more about Step one B 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 D n a lie Gaze is an enzyme, and it is an enzyme that lie. Gates or CO. Valiantly joins the two sticky end together that were created in Step one A and that is going to create the final recombinant DNA molecule that contains DNA from two 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 litigated 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 litigated together. And so if we take a look at our example down below, we can see that a restriction enzyme and a DNA delegates are both needed, uh, and used to clone a recombinant DNA plasma and so down below. Here we're looking at creating recombinant d n A. And so it's important to note is that over here on the left hand side, we're showing you a bacterial plasmid over here 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 it's important to note Is that of course, in step one A. We know that restriction enzymes are going to be used, And, uh, there's the restriction. Enzymes are going to recognize restriction sites. And so over here, zooming into the plasma DNA, you can see that there's one restriction site, as you can see, and over here in this gene of interest, uh, notice that it is being flanked by two restriction sites, okay. And the actual gene of interest is just a 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 gene of interest. And as we know from step one A from our previous videos using restriction enzymes 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, uh, 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. Uh, now, uh, being pieced back together with right in between the d n. A plasma where the DNA plasmid was cut. And so, of course, in order to co valiantly join and seal the gene of interest in the middle with the plasma, it, uh, what we'll need is these Legatus Enzymes and the Legatus enzymes are being represented by these little glue bottles because the DNA like this 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 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 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 recombinant DNA molecule would be made, we can now talk about the transformation process getting this recombinant DNA molecule into a bacterial host cell. But for now, this year concludes our, uh, introduction here to step one, be using ligation enzymes, and we'll be able to get some practice applying these concepts as we move forward in our course