Biology: Detect DNA Homology-Biotechnology Summary

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One of the greatest things about being a lecturer is you have a captive audience, and you can tell people about the research you've done. And I'm excited right now, so please don't click off because I want to tell you about the kind of work we're doing in my lab, but that's not just to brag, maybe just a little. You're about to see a compilation of all of the genetic techniques that would happen in a typical research project. Let me set the stage.
It's been known for a while that there's a very bizarre mutation in fruit flies, and this mutation is called antennae pedia, and this antennae pedia mutation is unbelievable because it causes the growth of legs on the head of a fly instead of antennae. That's phenomenal because that suggests that, and one thing I've got to tell you, it's a trait that can be passed on, so here's the thing. It suggests that it's a single trait, but you and I know enough about genetics to know that what controls a leg must be more than one protein, must be more than one gene. So if it's passed on like one gene, and it is one gene, it must be a very important gene. There's more on this.
There's a wasp, this is in fruit flies, Drosophila, there's a wasp we work with called Nasonia, genus name, and Nasonia has the same mutation, antennae pedia. And sure enough, this antennae pedia gene causes legs on the head of the Nasonia. Now, other insects have this, too. So what we're trying to figure out is, could this antennae pedia gene be perfectly the same as that in Drosophila? Is there DNA homology? Is the AGCT, AGCT sequence the same in Nasonia as in Drosophila, which is exciting, because if it is, it means that this gene has been conserved throughout the course of the hundreds of millions of years of evolution that wasps and flies have diverged. And if it's the same in them, might you or I have this gene, and we don't know what it does. Well, there's a lot to this question, which we're not going to get into now because it has to do with the development of genes. But let's take a look at how we might attack this problem.
Well, the first thing we need to do is we need to get the gene. We need an antennae pedia gene. And this is where communication with scientists is crucial. It's already well documented, and it's even sequenced in fruit flies. So the first thing I had to do was call a lab that has worked on antennae pedia. So we called Kaufman Labs at the University of Indiana, and here's what we were sent. We were sent a bacterium. Now a bacterium has nothing to do with fruit flies except for one thing. In the bacterium was a plasmid. In the plasmid was a gene. What was that gene? The antennae pedia gene. In other words, for my lab, we borrowed a bacterium from a gene library. And this was given to us, and we grew up this bacterium.
So step one was we got the bacteria. The second step was, now we need to get the plasmid out, so we take out the plasmid. And the third thing was, we have to cut that plasmid out. Well, when you order a plasmid from somewhere or someone, you get a map with that plasmid. If this sounds like a fairy tale, it's not. And with the plasmid came a map, and guess what. We were told the cuff sites on either side of the gene we wanted. So we were able to add restriction enzymes and cut that out. So what happens next? Are you with me so far?
So now, I'm cutting out the antennae pedia gene from right here, actually a segment of the antennae pedia gene, but that's just between you and I. So we cut out a segment of the antennae pedia gene, and what do I have now? Now, after growing my bacteria up, and that plasmid came with ampicillin resistance on it so I could grow my bacteria in a plate, and I knew they were my bacteria, actually not even in a plate, I grew them in a vial with nutrients in it. I then took the plasmids out, and I cut them. So now I have a test tube with broken up plasmids, and the plasmids have the regular bacterial plasmid DNA and the section of the gene called antennae pedia. Now I have to separate them.
How would you recommend I separate them? Gel electrophoresis. So, what we do is we run the plasmid in a well, and the big chunk goes down up here, and the small chunk with the plasmid on it is right here. We take out that high-faluting thing called a razor blade, we cut this out, and we dissolve the agarose. Now, we have a test tube, and what's in that test tube? Double-stranded antennae pedia gene. What are we going to use this for? We're going to use this as my probe. So what I have to do now, because remember, what is this? This comes from the fruit fly. We know the sequence of this. We want to know if this gene is on my wasp. So this is my known. This is going to be my probe. So it comes from the fly, and we have to now label it. So what we're going to do is we're going to single strand that and label. So now we have the antennae pedia gene. The piece I used was about 1000 base pairs long, and it's hot. It's labeled, it's single stranded, and it's ready to go. Where's it going to go? Well, there's more to do.
So remember, here's my hot probe. We'll put that over there because we're going to come back to that later, sitting in the test tube. But now here's what we have to do. What we have to do next is we have to get our wasps. So we're going to take our wasps, and we're going to grind them up. And we grind them up in liquid nitrogen, which is always fun, and we extract their DNA. So we get our wasps, and we do what is called a DNA extraction, and from that we get a whole piece. We call it genomic DNA, all of the wasp's DNA. Because what am I hoping? I'm hoping that somewhere in that DNA is what? Somewhere in that DNA is the antennae pedia gene.
Just a little bit on experimental procedure. I also better grind up some flies. Why? Because what do I know? I know that somewhere in the flies is the antennae pedia DNA. That's my what we call what? That's called a control group. That's my positive control group. So now I've got their DNA. What would you do next?
If I take that DNA and run it in a gel, all I'm going to get is a hunk of DNA. The DNA will stay in its entirety, and you'll just get this "bluph," and it'll just move a little bit because it's too much. Is there a way to make that DNA smaller? You bet. I use enzymes. So we cut it with restriction enzymes. Everything comes together in this project, enzymes, the same restriction enzyme here, and now I put it in there. Now, how many places do you think an entire genome is going to cut with one restriction enzyme? In millions of places. There are billions of nucleotides, and it's going to cut in thousands or millions of places. So you're not going to get those nice clear bands I've shown you. You're going to get, in essence, a smear. That smear represents maybe 10,000,000 bands. But nevertheless, it's still bands. This is my wasp. This is my fly. So now I want to get this probe on here. What do I do?
You know what to do. We need to do a Southern blot. So we're going to take this gel, and we're going to run it. Now, I've only shown you two runs. We actually ran about eight because there are a lot of different things you do, and there's my smear. To me, that looks like a smear. And I'm going to take that, and I am going to run a Southern blot on this thing. Remember about Southern blots? What we do is we put the gel in a denaturing solution, and now the denaturing solution is going to soak up, and it's going to take that DNA, my wasp DNA, and it's going to single strand it. And it's going to take my fly DNA, and it's going to single strand that. And what's it going to deposit it on? It's going to deposit it on a nylon membrane, a membrane that I can then peel off of that gel, and my DNA will be stuck to that membrane. And now I'm almost done because now that I've taken the DNA and put it on the paper, I can now use my probe.
So I get a bag. I put my nylon membrane with my hopeful DNA in there. Somewhere on here, it's invisible, but somewhere on there, we're hoping my DNA is. We seal the bag, but first we add our probe and a liquid. We seal the bag, we put it at a certain temperature, and we leave it overnight. The next day we come in, we take the bag out, we open it, and we take out the nylon membrane. We wash it. What does that do? It removes all probe except the probe that has hybridized. Why is it hybridized? We can only hope it's hybridized because the sequences are all the same or close. And guess what. Sure enough we found out that there were sections on that wasp that lit up. Would you expect it to light up on the fruit fly, too? If it doesn't, you have no data because we know it had to be in the fruit fly. What does that show? It shows that there is homology somewhere between the antennae pedia of the wasp and the antennae pedia of the Nasonia. What's next?
Well, where is this gene? What does it do? Is it present in other insects? Is it present in you? And if we think about that, now we start to see how we can trace an evolutionary lineage because now if we could sequence this gene, maybe it's just off by two or three nucleotides. And maybe caterpillars will be off by 10. And maybe humans will be off 20. But nevertheless, it shows an evolutionary ancestry.
The whole field of biotechnology, all of you who go into biology, you will learn biotechnology whether you are ecologists, whether you are biostatisticians. Biotechnology is the future for biologists.
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