List of restriction enzyme cutting sites: S

Transcription

- [Voiceover] All right, so in this video, we're gonna be talking about something known as restriction enzymes. Now, what are restriction enzymes? Well, let's go through an example, and hopefully that'll help answer that question. So let's imagine that we've got a bacteria. And it's just floating around, it's doing its thing, and out of nowhere comes this virus. And the virus is just kinda upset today, so it decides that it wants to infect this bacteria. So, what the virus does is it goes over to the bacteria, and basically it attaches to it, and it injects viral DNA into the bacteria. So this is the viral DNA, and I'll just label that here, so viral DNA. And the bacteria, being a living creature, also has its own DNA. So this right here in the blue, is the bacterial DNA, and I'll just label that here. So bacterial DNA. So basically, the virus infected the bacteria. But the bacteria wants to figure out some way to destroy the viral DNA. And one way that it can do that, is by labeling its own DNA. So let's imagine that the bacteria labels its DNA with these purple dots. So what these purple dots actually are, is a methyl group. So we'll say that these purple dots are methyl groups. And in order for the bacterial DNA to be methylated, there's an enzyme known as methylase. So methylase is an enzyme that basically floats around, and as bacterial DNA is synthesized, this enzyme methylase goes around and methylates bacterial DNA. Now, what this basically does, is it allows the bacteria to recognize its own DNA, and recognize any DNA that's not methylated as foreign DNA. So what we now have is another enzyme that's floating around in the cytoplasm of the bacteria. And that enzyme is known as a restriction enzyme. So this restriction enzyme. So this restriction enzyme is kind of floating around, it's doing its thing, and it recognizes the methylated bacterial DNA, but then it sees this foreign, unmethylated DNA, and it goes and destroys it. So this is basically a way for bacteria to protect itself from being infected by viruses. And it basically does so by methylating its own DNA, and destroying any other foreign DNA that is unmethylated. So the reason that this is called a restriction enzyme, is because researchers were noticing that certain bacteria were actually, they were resistant to being infected by viruses. So they were basically restricting viral growth. Thus, when they figured out what enzyme is causing that, they basically called it a restriction enzyme, 'cause it restricted the growth of viruses. Now, in order for the restriction enzyme to work, it needs to recognize a specific sequence, a specific bacterial sequence. So let's go ahead and write out an examaple sequence. So imagine that, if we zoomed down to the nucleotide sequence of the bacterial DNA, let's imagine that maybe the sequence was G-A-A-T-T-C. And of course, if this was one strand, then the sister strand would be G-A-A-T-T-C. Now, I don't know if you notice, but there's something unique about this specific sequence. So if you read this top line from left to right, G-A-A-T-T-C, and you read this bottom strand from right to left, G-A-A-T-T-C, they're basically identical. And this is known as a palindromic sequence. So palindromic sequence. Now, basically what a palindromic sequence is, is exactly this and it's what restriction enzymes recognize. So let's imagine that we have a restriction enzyme, we'll just give it a name, let's imagine that its name is EcoR1, so EcoR1. That's the name of one type of restriction enzyme. EcoR1 is actually able to recognize this palindromic sequence. So as EcoR1 is floating around the bacterial cell, it'll recognize this sequence, and if it's methylated, it won't touch it. But if it's unmethylated, as in the viral DNA, it'll actually cleave it, so it'll come here, and it'll cleave it. Let me actually use a different color. So it'll cleave the DNA. And it'll cut it like so. And what that'll do is it'll result in two strands. Because it just cut the DNA. So now what we're gonna have is we're gonna have one strand that's gonna be G, and then this bottom strand, T-T-A-A, and then we're gonna have this top part over here. So we're gonna have an A-A-T-T-C, and then a G down here. So basically, now these two strands are just floating around, and these ends down here, so this end kinda hanging over the edge, they're known as sticky ends. So these are known as sticky ends. And the sticky ends basically will float around, and if they contact one another, since they're complementary strands, they'll just reanneal. So normally if this gets cut, then you'll have two sticky ends, they'll float around and then they'll reanneal. And we can actually take advantage of the fact that we can have the strands reanneal, and we'll talk about different ways that we can actually use that for medicinal purposes. So one way that we can use this to our advantage, would be, let's say that we wanna synthesize human insulin. So let's say that I wanna make human insulin. So how can I make human insulin using the restriction enzyme technology? Well, let's imagine that we have a bacteria over here. And this bacteria has its bacterial DNA inside. And what we can basically do, is take this bacterial DNA, so let's extract it out of the cell, so we'll move it out here. So here's the bacterial DNA. What we'll do is we'll take EcoR1, and we'll basically cleave the bacterial DNA at this site. So let's say that we cut it right here. So if we cut it right there and then we kinda unroll the bacterial DNA, now we have this space. And this space basically has these sticky ends here. So I'll just fill those in. So let's say that there's a G C-T-T-A-A, and then over here on this side, I don't know if I have enough space, we'll have an A-A-T-T-C and a G. So now we've got these sticky ends over here. And what we can go ahead and do is we can take the human insulin gene. So we'll take human insulin, so this is the insulin gene. And we'll have exposed it to EcoR1 already. So now it's got the sticky ends over here. So now on this side we've got an A-A-T-T-C and a G, and then over here on this side we've got a G, and a C-T-T-A-A. So now what we can do is we can take this whole thing and just plop it right in here. And as you can see, this part of the insulin gene will reanneal with this right here. And then this part of the insulin gene will reanneal with this part of the bacterial DNA. And basically now what we have is we have the insulin gene inserted into the bacterial DNA. And what that'll basically allow the bacteria to do is it'll allow it to synthesize human insulin. And now what you do is you just take that insulin and you purify it, and now you can basically have a whole bunch of insulin that's made very cheaply and very quickly for diabetic patients that might need the insulin. So that's one example of how restriction enzymes can be used in the pharmaceutical and biotechnological world.