22. Techniques in Cell Biology
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Hello everyone in this lesson. We are going to be talking about D. N. A. Cloning. Okay so some of you in the future if you do biology lab or some sort of experiment you may want to do DNA cloning. And I don't mean we're cloning an entire organism. I don't mean we're making a new dolly or something like that. I'm talking about cloning small segments of target D. N. A. Target D. N. A. Is basically any gene or set segment of DNA that you are interested in and you want to study. So the first thing to do whenever you're trying to clone a segment of D. N. A is you want to obtain that particular piece of DNA. So that's the first thing you want to do. And generally the way you're going to do that is you're going to take the organism, you're going to take a couple of cells from the organism of interest. And then you're going to cut that segment of DNA out. You might create multiple copies of that segment of DNA via the preliminaries chain reaction for the specific sequences. And then once you have your sequence and you want to clone it, the next thing you're going to do is you're going to digest your segment of DNA with restriction enzymes. And this is going to cut the D. N. A. Into the very unique sequences that you want to clone a restriction indo nucleus is going to be a very important type of protein that basically cuts the D. N. A. And it's going to cut a segment of DNA out now they are going to cut segments of DNA out but they're not going to do it randomly. They're going to cut at the restriction sites. So there are many different types of restriction indo nuclear bases or restriction enzymes. And each one of them has their own unique restriction site that they recognize. So it may be a particular sequence, maybe it's A G. G. C. T. A. And whenever that particular restriction enzyme sees that sequence in the D. N. A, it's going to cut it right there. So you may look at your piece of D. N. A. And you think I want this 10 base pair segment. How do I cut it out of there? Well what are the sequences where I wanted to cut? Once you figure out the sequence where you wanted to cut you find a restriction enzyme that cuts at that sequence. So it's a very specific protein and it's very useful type of protein in DNA work and genetic work. It can cut D. N. A. It can also cut RNA. There are very unique restriction enzymes. So what you're going to do is your you're gonna get your sequence of D. N. A. And then you're going to cut your desired gene with restriction enzymes out of that segment of D. N. A. And then the next thing that you're going to do is you're going to paste it into the desired vector. This is basically a cut and paste scenario You cut out the sequence that you want and you're going to paste it into a new sequence. So pasting isn't as easy as it may sound. You're going to pay taste your unique piece of DNA that you just cut out with a restriction enzyme into something called a vector. Which is also commonly known as a plasmid. You're generally going to find these in precarious plasmids are going to be small rings of D. N. A. That are found inside of pro carry arctic organisms that are not the chromosome of that pro carry attic organism. So basically it's like a tiny segment of D. N. A. That is not the chromosome but it's still replicated with that pro carry out. So what you're gonna do is you're going to take your segment of D. N. A. That you just cut out and you're going to paste it into this plasma or this vector. And it's important to know that plasma is replicate independently of their organism and of the chromosome of that organism. Now, whenever you cut open a plasmid to place your segment of D. N. A. Into that plasma, you're going to want to cut the plasmid with the same restriction enzyme that you cut the D. N. A. With to ensure that the ends of the D. N. A. And the ends of the plasma are able to complementary lee bind with one another. So let me write that down for you guys. You want to cut the plasmid with the same enzyme and this is going to ensure that the ends of the plasma and the ends of your segment of D. N. A. Of interest are able to bind with one another. Now once you are able to actually get those two pieces of DNA in place, you're going to bind them together or glue them together with DNA. Like remember we learned about DNA legs whenever we were learning about DNA replication that happens naturally in cells. And DNA lig, his main job is to glue segments of double stranded DNA back together. We're going to use it for the same purpose here. But instead of gluing different segments of DNA back together, we're going to glue our segment of D. N. A. Into the vector or into the plasmid. So it's going to seal those two DNA fragments together. And now we have something called a recombination plasmid. It's going to be a plan that has the original DNA from the precarious plus the sequence of DNA that we want to clone. So now what we're gonna do is we're going to place the vector containing the D. N. A. Sequence into an organism and this is generally going to be E coli E coli is very very very commonly used for cloning. It does a really good job. So every time the E coli replicates the plasma are going to replicate and you're going to clone a whole bunch of those plasmids or those vectors and you're going to clone your segment of DNA many many times. So it's going to allow for incredibly large quantities of cloning of the specific DNA sequence. This is the way that we clone DNA into the largest number of clones that we are able to do, you may be thinking why would we want to do this? Well, if the sequence of DNA is incredibly important, maybe it's a gene for a particular disease. And many scientists want to work on that gene, they each get a certain number of copies of the clone of that gene. So you're going to need to replicate it or clone that gene a lot. This cloning method is also utilized when you're studying the gene or when you want to do a transgenic experiment or create a transgenic organism, these kinds of things. Also this is important for gene therapy. You have to clone the gene a lot for gene therapy. So basically this is just utilized to better understand this particular segment of DNA and to use it in experiments. Okay, alright, so now let's have a look at our figure. Okay I'm gonna go out of the figure because obviously I'm in the way but I'll come back at the end. Okay so this is just going to be a diagram of exactly what I already talked about. So we have this sequence of original D. N. A. From the original organism. So this is our organism of interest. Maybe it's a human being, maybe it's some other organism but generally it's going to be probably human gene that we want to clone. So we can better understand that particular gene. And then we're gonna have our restriction enzyme right here and our restriction enzyme is going to locate its restriction sites and it's going to cut at those restriction sites. So it's going to cut right here and it's going to cut right here. And that is going to create this segment of D. N. A. Which is no longer bound to the other sequences of DNA. So this is generally called our target sequence sequence. This is gonna be the sequence of D. N. A. That we are interested in and that we particularly want to clone. Now, remember I told you that the restriction enzyme also needs to cut the vector wherever it wants to insert that particular sequence of D. N. A. So the vector is going to be cut with the same restriction enzyme as the original organisms. D. N. A. Was cut. So then we're going to insert that segment of D. N. A. Into the vector where the restriction enzyme has cut. And then Legacy is going to seal it up. So let's rate that Legacy glues glues target sequence to the vector glues target to the vector and you guys can see here that it is all glued together and that it is one plasmid or one vector. So this is commonly called a vector and you guys can see that they've placed this particular vector into the E. Coli bacteria. Now this vector is going to be independent of the chromosome of the E. Coli. So let's say that this is the chromosome of the E. Coli. The vector is not a component of D. N. A. Of the chromosome. It is going to be an independently replicating segment of DNA that is not connected to the chromosome. And then that vector and the E. Coli are going to replicate themselves and you'll eventually have all of these different E. Coli with all of these vector with your D. N. A segment in them. So you're going to have a ton of vectors with this D. N. A segment. So you're going to have a ton of replicated or cloned segments of your target sequence. And then basically all you do is you remove the vectors from the E. Coli and you're going to actually cut your target sequence out of the vector and then you're going to have, gosh knows how many, probably a ton of your target sequence. So this is going to be the basic way that we clone massive amounts of D. N. A. At the moment in time. This is going to be the main way that we do this. You guys may even do this in an experiment or in an upcoming lab. I hope that this was helpful. Let's now go on to our next lesson.
Which of the following shows the correct order of DNA cloning steps?
DNA extraction → Digestion → Place DNA into E. coli → DNA ligase
DNA ligase → Digestion → DNA extraction → Place DNA into E. coli
DNA extraction → Digestion → DNA ligase → Place DNA into E. coli
Digestion → DNA extraction → DNA ligase → Place DNA into E. coli