Steps to DNA Cloning

in this video, we're going to talk about the steps to DNA cloning. And so recall there are two general steps to DNA cloning and we have them down below label number one and number two. And so the first step in DNA cloning is just making or creating the recombinant DNA molecule. And the second step is going to be transforming the DNA molecule, which recall just means allowing bacteria to uptake the recombinant DNA. And so, if we take a look at our image down below, we can get a better understanding of these two steps involved with DNA cloning. And so, over here on the far left, what we have is the first step to DNA cloning, which is creating the recombinant D n A. And recall that recombinant D N A is a single molecule that contains DNA from two different sources. And so here in this image, what we have is our bacterial plasmid over here and green, and we have over here our gene of interest, which would be from a different species like, for example, a human. And so here this gene of interest is a gene coding for some protein that we're just calling protein X here, Okay. And so 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 d n A. And so you can see these little scissors here that we're using as just a symbolic representation, uh, to show you the cutting that must occur in order to create the recombinant DNA molecule. Now in reality, doesn't actually use scissors. Okay? 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. And so the first step is going to be the cut. The d n A. And after the d. N A has been cut. As you see here, the second part here, part one b is going to be to litigate the d n A and litigate the d. N. A is basically like pasting the DNA together, so it's almost like a cut and paste kind of thing. One a. Here is cutting the d. N. A. And one B is litigating the d n A or pacing the DNA together. So you can see we've got these little tiny glue bottles here just, uh, just being used as a symbolic representation of DNA Ligue aces, which are going to be the enzymes that litigate or pace together or seal together these two DNA molecules. And so you've got, uh, these cut fragments over here that are going to be pasted together into a single molecule. So you've got the bacterial plasmid is over here, and the gene of interest coding for protein X is within the same molecule. And so then this, um, 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 two is transformation of the recombinant DNA. And so, of course, transformation here in this constant contact is just talking about allowing the bacterial cell, which is right here to uptake the external d n A. And so it's going to uptake the external d n A. And be able to obtain that recombinant DNA molecule. And so notice that the scientists way over here is now saying, uh, now that he's got this recombinant DNA molecule within the bacterial hostal. He's saying he can grow this bacteria and allow the bacteria to express as much protein X as he needs. And so, basically, through DNA cloning, uh, we are able to clone the D N A and also create, uh, as much of the gene of interest, the product of the gene of interest that we're trying to create. And so this is something that we're going to continue to talk about more and more these two steps, Uh, step number one, 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. And so this is just the introduction here. And so that concludes this introduction to the steps to DNA cloning. And 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

in this video, we're going to talk more details about step number one of DNA cloning, which is creating the recombinant DNA and so creating a recombinant DNA molecule is actually a two step process within itself that requires Step one A and Step one Be now Step one A is using restriction enzymes in order to cut the DNA. And so these restriction enzymes, they kind of are like little tiny molecular scissors in a way, because what they do is they can cut the DNA molecule. And so you can see that we're using these scissors in the image to represent the restriction enzymes. And so the restriction enzymes are going to cut the d N A. Now after the d. N A. Has been cut. The second step, part one B, is going to be to use Ligue ace enzymes. And lying is enzymes are going to be enzymes that are going to paste the D N A. Essentially, it's going to act like glue in a way, And so over here, what we have is a little bottle of glue, and that little bottle of glue is supposed to be the symbolic representation of Legatus Enzymes or enzyme that are going to seal together and co violently linked the cut DNA molecules. And so you can see that here we've got the cut bacterial plasmid and the cut gene of interest that are being pasted together by the league enzymes, which again are represented by these little glue bottles that you see here. And so at this point, what we have is the final result, which is the recombinant d n A. And that concludes step number one, the creation of recombinant DNA. And so, um, this here concludes this video, and we'll be able to move on to step number two in our next video.

in this video, we're going to talk more details about step one A in the steps of DNA cloning, which is using restriction enzymes and so recall that restriction enzymes are enzymes themselves that are specifically important for cleaving or cutting the d. N. A. At these specific locations called restriction sites on the d n A. And that is going to produce sticky ends. And so we're going to define restriction sites and sticky ends down below in our text. And so restriction sites are really just defined as specific sequences of DNA where a restriction enzyme will bind and cut the DNA. And so these restriction enzymes, they don't 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 regions of the DNA called restriction sites. And, 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 d n, a overhang that is going to be produced from restriction, digestion reaction and so we can get a better feel for this down below in our image, looking at restriction enzymes and restriction sites along with sticky ends. And so notice over here on the left hand side, what we have is a specific D n a, uh, 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 ends up. And 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 the restriction site. Now, 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 d. N A. And so notice that the D. N A. 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, um, are kind of, uh, sticking out of the rest of the molecule. And so these are these sticky ends that we are referring to, and the reason that they're called sticky ends is because they can still complementary base pair to other matching sticky ends as well. Talk about moving forward in our course, but for now, this year concludes our brief lesson on Step one a. Using restriction, enzymes and how restriction Enzymes will bind and cut restriction sites to generate sticky ends and, uh, separate molecules. And so we'll be able to get some practice applying these concepts and talk more about Step one be as we move forward, so I'll see you all in our next video.

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

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 too 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, 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 Fiona typically 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 Leganes to litigate or join them together to create the recombinant DNA molecule, which is going to have the gene of interest, uh, connected to the bacterial plasma 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 up taking external DNA like this cloning vector here and so through the process of transformation, noticed 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 of 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 Nicola. And, uh, now it has antibiotic resistance that now that it has received the gene of interest with the antibiotic resistance, and so these, uh, this transformed bacterial cells. They can actually replicate and express the gene of interest, and the researcher can then purify the jeans product and study the gene of interest in the product of the gene of interest. And so this here is really the conclusion to the second step of DNA cloning, which is transforming 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.

in this video, we're going to do a review and talk about the application of DNA, cloning and 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 of medicine. Now, diabetics actually do not produce enough insulin protein to metabolize blood glucose. And these diabetics, they require daily injections of insulin. Now, researchers have found a way to use transgenic organisms, uh, 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, uh, 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 legs, 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 plasma is just going to replicate via its normal replication process, creating a bunch of bacteria. And each of these bacteria are going to have a copy of the recumbent 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 producing the human insulin gene, and the cloned insulin genes can be used for other experience and the human insulin hormone, the protein that's being expressed, that's created by the bacteria. It can also be, uh, 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, Uh, and so this year, concludes our brief introduction to the application of DNA cloning and 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.