DNA cloning and recombinant DNA
- [Voiceover] Let's talk a little bit about DNA Cloning. Which is all about making identical copies of a piece of DNA. And usually it's a piece of DNA that codes for something we care about, it is a gene that will express itself as a protein that we think is useful in some way. Now you might have also heard the term cloning in terms of the Clone Wars in Star Wars or Dolly the sheep and that is a related idea. If you're cloning an animal or an organism, like a sheep, well then you are creating an animal that has the exact genetic material as the original animal. But when we talk about cloning and DNA cloning we're talking about something a little bit simpler. Although, as we'll see, it's still quite fascinating. It's identical copies of a piece of DNA. So how do we do that? Well let's say that this is a strand of DNA right over here and I'm just drawing it as a long, but this is a double-stranded, and I'll just write it down, this is double stranded. I don't want to have to take the trouble of keep drawing the multiple strands. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves. So there we go. This is the double-stranded DNA and let's say that this part of this DNA has a gene that we want to clone. We wanna make copies of this right over here. So gene to clone. Gene to clone. Well, the first thing we wanna do is we wanna cut this gene out some how. And the way we do that is using restriction enzymes. And there's a bunch of restriction enzymes, and I personally find it fascinating that we as a civilization have gotten to the point that we can find and identify these enzymes and we know at what points of DNA that they can cut. They recognize specific sequences and then we can figure out well which restriction enzyme should we use to cut out different pieces of DNA, but we have gotten to that point as a civilization. So we use restriction enzymes. We might use one restriction enzyme, Let me use a different color here, that latches on right over here and identifies the genetic sequence right over here and cuts right in the right place. So that might be a restriction enzyme right over there and then you might use another restriction enzyme that identifies with the sequence at the other side that we wanna cut. So let me label these. These, those things right over there those are restriction enzymes. Restriction enzymes. And so now you would have, after you applied the restriction enzymes, you will have just that gene. You might have a little bit left over on either side but essentially you have cut out the gene. You've used the restriction enzymes to cut out your gene and then what you wanna do is you wanna paste it into what we'll call a plasmid. And a plasmid is a piece of genetic material that sits outside of chromosomes but it can reproduce along, or I guess we can say can replicate along with the machinery of the, the genetic machinery of the organism. Or it can even express itself just like the genes of the organism that are in the chromosomes, express themselves. So then so this is where we cut, let me write this, we cut out the gene and then we wanna paste it then we wanna paste it into a plasmid. And plasmids tend to be circular DNA so we will paste it into a plasmid. And in order for them to fit there's oftentimes these overhangs over here. So you might have an overhang over there, you might have an overhang over there. And so the plasmid that we're placing in might have complimentary base pairs over the overhangs, which will allow it easier, it will become easier for them to react with each other if they have these overhangs. So let me, we're pasting it into the plasmid. And this is amazing because obviously DNA, this isn't stuff that we can, you know, manipulate with our hands the way that we would copy and paste things with tape. You're making these solutions and you're applying the restriction enzymes. The restriction enzymes are just in mass cutting these things. They're bumping in just the right way to cause this reaction to happen then you're taking those genes and you're putting them with the plasmids that happen to have the right sequences at their ends so that they match up and then you also put in a bunch of DNA ligase. DNA ligase, to connect the backbones right over here. And we also saw DNA ligase when we studied replication. So that is DNA ligase, which you can think of it as helping to do, helping to do the pasting. And so now we have this plasmid and we want to insert it into an organism that can make the copies for us. And an organism that's typically used, or a type of organism is bacteria and E. coli in particular, and so what we could do is, we could, let's say that we have a bunch, let's say you have a vial right over here. You have a vial and it has a solution in it with a bunch of E. Coli. A bunch of E. coli. And you actually wouldn't be able to see it visually but there is E. coli in that solution. And then you would put your plasmids, which would be even harder to see, in that solution and somehow we want the E. coli, we want the bacteria to take up the plasmid. And the technique that's typically done is giving some type of a shock to the system that makes the bacteria take up the plasmids. And the typical shock is a heat shock. And this isn't fully understood how the heat shock works but it does and so people have been using this for some time. So if you have a bacteria, you have a bacteria right over here, it has its existing DNA, so this is its existing genetic material right over there, let me label this. This is the bacteria. You put it in the presence of our plasmids so you put it in the presence of our plasmid and you apply the heat shock and some of that bacteria is going to take in the plasmid. It's going to take in the plasmid. And so just like that, it's going to take it, it's going to take it in. And so what you then do is you place the solution that has your bacteria, some of which will have taken up the plasmid, and you put it and then you try to grow the bacteria on a plate. So let me draw that. So let me draw, so here we have a plate to grow our bacteria on, and it has nutrients right over here that bacteria can grow on. It has nutrients. It has nutrients, and so you could say, okay well put this here and then a bunch of bacteria will just grow. So you would see things like this, which would be many, many, many cells of bacteria, there would be colonies of bacteria. You could just let them grow but there's a problem here. Because I mentioned some of the bacteria will take up the plasmids and some won't. And so you won't know, hey when this bacteria, when it keeps replicating it might form one of these, it might form one of these colonies. So this is a colony that you like. So this one is a good colony, put a checkmark there. But maybe this colony is formed by an initial bacteria or a set of bacteria that did not take up the plasmid so it won't contain the actual gene in question. So you don't want that one. So how do you select for the bacteria that actually took up the plasmid? Well, what you do is besides the gene that you care about that you want to make copies of, you also place a gene for antibiotic resistance in your plasmid. So now you have a gene for antibiotic resistance here, and so only the bacteria, and I think it's amazing that we as humanity are able to do these types of things, but now only the bacteria that have taken up the plasmid will have that antibiotic resistance. And so what you do is in your nutrients you grew nutrients plus antibiotics, plus an antibiotic. Antibiotic, and so this one will survive 'cause it has that resistance. It has that gene that allows it to not be susceptible to the antibiotics. But these are not going to survive. They're not even going to happen. They're not even going to grow because there's antibiotics mixed in with those nutrients. And so this is a pretty cool thing. You started with the gene that you cared about, you cut and pasted it into our plasmid. Let me write the labels down, into our plasmid that also contained a gene that gave antibiotic resistance to any bacteria that takes up the plasmid. You put these plasmids in the presence of the bacteria or you provide some type of a shock, maybe a heat shock, so that some of the bacteria takes it up and then the bacteria starts reproducing. And as it reproduces it also is reproducing the plasmids and because it has this antibiotic resistance it is going to grow on this nutrient antibiotic mixture and the other bacteria that did not take up the plasmids are not going to grow. And so just like that you can take this, you can take this colony right over here, and put it into another solution or continue to grow it and you will have multiple copies of that gene that are inside of that bacteria. Now the next question, and I'm over simplifying things fairly dramatically is well you now have a bunch of bacteria that have a bunch of copies of that gene, how do you make use of it? Well, the bacteria themselves, let's say that gene is for something you want to manufacture say insulin for diabetics, well you could actually use that bacteria's machinery, we used its reproductive machinery to keep replicating the genetic information, but you can also use its productive machinery, I guess you could say, it's going to express its existing DNA but it can also express the genes that are on the plasmid. And in fact that's what would give the bacteria its antibiotic resistance but if this gene was say for insulin, well then the bacteria will produce a bunch of insulin, a bunch of insulin molecules, which you might be able to use in some way. And I'm not going to go into all the details of how you will get the insulin out and how you could make use of it, but needless to say, it's pretty cool that we can even get to this point.
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