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This lecture explores fundamental molecular biology techniques, focusing on transcription, translation, and gene expression. It delves into the processes of dna replication, pcr, and reverse transcriptase, highlighting their significance in research and applications. The lecture also discusses the use of different vectors and promoters for gene expression, emphasizing the importance of understanding these techniques for studying gene function and developing new therapies.
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How are we doing? Speaker 2 - 00: Yes. Oh, it's all right. Speaker 1 - 00: I just had a long day. I started at 09:00 in the morning. Speaker 2 - 00: I didn't get here till after. Speaker 1 - 00: I was in here for 3 hours. Speaker 2 - 00: Oh, that'll do it. Which class did you have? Speaker 1 - 00: I had a doctor denning for the infectious disease seminar class, and then in the morning, I had neuroanatomy with doctor Bodner. Speaker 2 - 00: Yeah. Yeah.
Long day. Yeah. We'll see how long I last. Speaker 2 - 01: Makes me a little crazy, because I'm used to having a clock over there. There was always a clock over there. Speaker 1 - 01: I think that clock was broken for a while. Speaker 2 - 01: Working. Yeah, I kept resetting it, and then I find. Can you tell me more about this gene you discovered? Sure. It's responsible for the photoreceptor. It's responsible for detecting light for as well as movement of chloroplasts. Speaker 1 - 02: Is this for only c three plants? Speaker 2 - 02: No, it's in cp and c four. In fact. It's nothing. Speaker 1 - 02: Well, yeah, we don't do phototropism. Sometimes we do when it's really sunny.
I will go ahead and bring in a schedule so people can start signing up for the first presentations that you're being given, just so that you can work things around. At the end of the last class, I was talking about production of genomic libraries and how you're going to cut up the DNA with a restriction enzyme. You're going to clone those fragments into a vector, and each one of those individual clones is going to have represented within it a part of the genome to the exclusion of all the rest of the genome. So you can really focus in on what in that particular cell. So you'll see why that's so important when, especially prior to rapid sequencing, that we're going to be talking about later on. Genomic sequencing methods. Speaker 2 - 08: This was an absolutely essential process for discovery, and before there was a lot known and understood about what was present in the genomic at that time, it became clear that you had to go digging for these individual genes with very little in the way of knowledge about what to expect. You didn't have sequence information, you didn't have functional information. It's much easier now, as you'll see later on. So there are going to be high throughput methods that we'll be able to talk about more toward the end of the semester. We'll be talking a lot about some of the more recent and most recent methods that have developed for molecular biology toward the end. And that's going to be the reason for that, is that you'll begin to see how, first of all, these methods evolve from what we know already.
And also you'll get an appreciation, I hope, for the process by which these different mechanisms within the cell for transcription and regulation of transcription, for translation, for replication and various other processes, how those developed and why it is that something that we take for granted now, things like PCR, things like knowledge of individual genes within the human genome, for example. That that process, in some cases, what we could do in the weekend now took several years back, 100 years ago, when I was a graduate student. Speaker 2 - 09: So you'll start to see this process, and I'm not going to dwell too much on these methods after we've sort of covered them here, except for you to understand when you're looking at a piece of data, a gel, for example, showing the binding site for a particular restriction enzyme, or the presence or absence of a particular mutation, that these processes meant that, or understanding these processes meant that it was possible to go through this very long process and eventually come out with information about protein interactions and how that affected the ability for transcription to initiate. So, like I said, I was talking last time about genomic libraries, and so I want to continue on this thread talking about seeding complementary DNA, production and production of libraries. Let's see why this is so important. So, a cdna stands for complementary DNA.
You don't have to worry about the intergenic regions, which are really important and really interesting. But when you're focused on functional genes that are translated, this will give you a snapshot of the, in a given set of cells, okay, so, typically, you'll have potentially thousands of genes that are being expressed at different levels. And unlike a genomic library where you take a genome, and no matter how many copies of the genome you have, all of the pieces of DNA are represented essentially equally in your library here, you're going to have more copies of genes that have more rna corresponding to them, that are more highly expressed. So in your library, you'll have more copies of that versus something that is made at very low levels or maintained at very low levels within the cells. Speaker 2 - 14: And so you get a representation, even though it's crude and it's nothing, it's only semi quantitative, but it corresponds to the relative amount of that message that's present oftentimes, and not always corresponds to the relative expression levels, even the protein level, ultimately. So, in order to make a cdna library, like I said, you have to start off with the template, which is the messenger RNA and eukaryotic messengers. As you'll see in great detail as we go through later on, eukaryotic mRNA's not prokaryotic, but eukaryotic will have a poly tail at the three prime end of the messenger RNA. And that poly tail is critical both in terms of the function, including its ability to be translated, of that mRNA translated.
But it provides us with a nice tag at the three prime end of that mRNA, so that we can put on now a primer which will correspond to that poly tail. Now, why is that important? Remember when I talked about the D nickel and races? Those require a primer, very few exceptions. They require a starting point, that three prime hydroxyl group of something that's bound to that other strand, template strand, so that it can work its way off of that. We'll get into the details of that later on. The same is true of reverse transcriptase that uses, in this case, rna as a template to make a DNA strand. It has to have a starting point. Speaker 2 - 16: But the beauty is that if these all have it there, three prime end, Apollo tail, we can add Oligodt, a piece of DNA that's just a stretch of t's that will bind to that poly tail. And once it's bound, it's now got a three prime end at the end of those t's there, that will allow polymerase, our reverse transcriptase, to make that second string or first string of DNA. Okay, with me so far, you're going to learn all this in way more detail. We then need to get rid of the rna because we're going to make the second strand of DNA. And so what we'll use is a very specific type of rnase that's going to break down rna, but not just any rna.
You have an rna bit that hasn't been removed. There was nothing to come along and remove that. And so you have mostly double stranded DNA, but you have this little chunk of rna at that end. Okay, how do we get rid of that? Well, first of all, what we can do is we can clone this double stranded DNA into a vector. And once it's in a vector, the cell will treat it like its own DNA, like it's supposed to have it, and it sees a gap or a nick in the DNA as something that it needs to fix, and it will fix it. Okay, so what will happen is we can put this into a vector, but in order to do that, we need to have something that is going to give it a sticky end. How do we do that? Speaker 2 - 20: Well, we can add using another enzyme, terminal transferase, which is a really unique enzyme that's going to be able to add nucleotides to the three prime end again of the DNA. But this is one of those very few examples of an enzyme of a polymerase that doesn't require a template. It's simply going to put a string of, if you only give it deoxy c, it will put a string of C's at this three prime end and at this three prime end. And you can do the same thing with the vector. You cut it at the multiple cloning site, for example, add terminal transfer ace, it will add, if you put in the oxygen g, it will put in a string of g's, the G's and the C's can now anneal. And you can just put that into the cell.
It will fix the backbone, it will fix any gaps that are present. And you'll get a circular piece of DNA containing your unit interest. But in addition, and they, it's a little strange here. Here they've just annealed it, but it hasn't actually finished repairing it yet. But the cell will recognize a nick here. It will fix this by removing the rna and replacing with DNA and fix the backbone. So you get a covalent now circle. Okay, so let me go through this a little bit differently. So if you're using an mRNA template, and in this case example that I was just talking about, you have a population of different mRNA's, but you could be doing the same thing with a single mRNA, a single transcript. Speaker 2 - 22: But if you have a population of these, what you're going to do is you're going to use your reverse transcriptase and you're going to do that using. So reverse transcriptase is an rna dependent DNA polymerase, rna dependent because it uses it in rna template DNA polymerase, it needs to have a primer and that primer has to have a three prime hydroxyl group. But it could be an rna or a DNA primer. You can use either one, which makes sense when you think about it, because once it started adding DNA to an rna, now you've got an rna end and it has to be able to respond to either one to add nucleotides to the three prime end of either one. Okay, so this poly tail works great because it's present in all mRNA's from eukaryotic systems. Okay, any questions?
The primer. Speaker 2 - 26: Yeah, well, the primer. Yeah, the primer. Do you mean because it's rna, you mean that it gets removed? Or do you mean because you need a primer to start with? Speaker 1 - 26: Because you usually would need a primer, like some sort of art primer to start, like, especially, like, in the lag. Speaker 2 - 26: And this is not lagging, or we'll get that with replication, but this is not lagging or leaving. This is a polymerize. Yeah, yeah, but you're absolutely right. So the problem is that when you have that rna, the rna can't be read, for example, by a ribosome. You're not going to get protein made. I'm sorry, what am I saying? You're not going to get for replication patient. You're not going to get the second strand made opposite the RNA. And furthermore, that RNA is always going to be more unstable and less likely to be 100% correct than the DNA, simply because of the way, as you'll see later on, with replication versus transcription, replication is much higher. It's called higher delegate. Exactly. Much less likely to have mistakes. And so it's very important that RNA be removed.
If it's simply removed, the problem is it's got to have something upstream of it that has a three pine hydroxyl group where it can fill in. You can't simply remove the rna or you just have a gap. You can't fill that in without a starting point upstream. You see why this is so important when we talk about replication. In particular, in eukaryotic systems, where you'll have typically linear chromosomes versus something like E. Coli, where it's circulating. Any questions so far? Okay, so like I said, there's this one enzyme. It's one of very few enzymes that are DNA polymerases that do not remain of require a template. All it needs is a starting point. It just needs a three prime hydroxyl group that you can add things to. But it doesn't have to have a template to read. Speaker 2 - 28: It's going to take whatever nucleotides, deoxynucleotides are present in the mixture. If you put in a mixture of deoxy, AGT and C, you'll get a mixture not quite random, it does have preferences, but still it will add nonsense or unpredictable sequences, I should say. But if you just put in one nucleotide, one deoxynucleotide, it will simply add a string of that nucleotide. And that's the power of using it in method I just talked about, because you're able to add what amounts to a sticky end that's predictable, and you can get it to match up with the vector where you do the same thing with the complementary nucleotide, the c on the, insert the g on the vector and those will come together and you'll be able to clone those. Okay, make sense?
But just to understand that, in order to make sure that you have the full length gene, that becomes really important, because if you're looking at regular sequences that are within the gene or within the untranslated region of the mRNA, if that's missing, you're going to miss that information and you're going to think that something is the beginning of the gene and it's not. So, in order to do this, it's possible to find the rest of the gene by using that now partial cdna as your starting point for as a, basically a primer, binding it to the mRNA. And that now you're starting instead of the poly tail, you're starting somewhere in the middle of the gene, and you're able to much more likely get to the five prime immune gene. Okay, there are methods. Speaker 2 - 32: Sometimes you'll also be missing the three prime end, but you wouldn't necessarily discover those, because if they don't have those three prime ends, they won't have a poly tail, which means the oligode won't do anything and you won't even know that mRNA exists. So the basic idea behind this is you've got your mRNA's and you've got a whole population of them. But your incomplete cdna, the one you're interested in here, is only going to bind to the complementary sequences. So most of the mRNA's it will ignore, but it will bind to its complementary template rna reverse transcriptase. Then, instead of starting back here and starting here, the three prime n is going to be able to complete that and get to this point.
And then again, you can use terminal transferase to add, in this case, c's, make the second strand using the polygon, it'll make the second strand here. And then you can produce a whole lot more of that product by PCR. I'll talk about pcr here in just a second. I know you're all familiar with that concept anyway, but we'll be using that a lot and talk about a lot of different modifications of PCR that are really valuable things which are not necessarily intuitive. It first, but ultimately you're going to have a product like this when you're dealing with this raised procedure, because you're using terminal transferase to again use rna, I mean, to produce the ends here and you're using reverse transcriptase to start into the mRNA further, so you're more likely to get to the five prime. Okay. Speaker 2 - 34: Another method, like I said, which I'll be talking more about later on, is instead of using terminal transferase to add these, it's possible to ensure that you're dealing with the actual five prime end of the gene by using a method known as rna ligase dependent. And the idea is that instead of adding the terminal transfer race this, which we'll add to any three prime m instead, what you do is on the rna itself. You make sure that you only are adding a known piece of rna or DNA to the end of the rna that's complete. And I'll talk about why that is when I talk about the structure of RNA. But the bottom line is you're going to have a g cap, it's called at the five prime end of that rna.
Primers will represent the ends of the DNA that you're amplifying. You'll see what I mean here in just a second. But you're going to have one primer that is going to be binding to the top strand. The other will bind to the bottom strand with their three prime ends facing each other, essentially. And that's going to allow me to make more of the product. And as you amplify these more, you're going to amplify, essentially, exponentially, specifically, this smaller fragment or defined fragment that is going to have what's present between or including those two primers and everything between them. I'm going to talk about this briefly just so you have, again, a good idea of this. It's going to be old news to most of you, but I just want to make sure you're clear on the process. Speaker 2 - 38: So the idea, when Kerry Mollus originally discovered this, he basically used normal polymerase. And the problem was that in order to make this work, we had to heat up the mixture so that the two strands of DNA would come separate from one another, so that they would be single stranded, so that then the primers would be able to bind. When you decrease the temperature again, you didn't have a cycler that allowed us to load our samples into the machine and walk away and go have dinner instead. Instead, he had to do this by moving things from one water bath to the next over and over again. And every time he did that with a normal polymerase, it would cause that polymerase at the high temperature to be destroyed. It would lose its functionality, which meant he would have to add polymerase each cycle.
And it was very slow, laborious, lots of ways for it to run into problems. We don't have to worry about that now. I won't get into that at this in a lot of detail now, but understand that you're starting off, in this case, in the first cycle, with a double stranded piece of DNA, which has undefined ends. It might be a piece of genome, whatever it is, and you throw in two primers, a forward and a reverse primer. You then see separate those two strands by heating it up to 94 degrees celsius or so. Cool it down to the annealing temperature. Depending on the sequence of these, they will bind. And you'll have so many more of these than of these strands that simply by probability, most of the time, it's the primer that's going to bind to each of these two strands. Speaker 2 - 40: So one you have that binds to this sequence with its three prime end here, the arrow, this one binds to this sequence with its three prime end here, those two new strands will be made. And now you have two new products which have one strand, the new strand, a defined end and an undefined end. So at this step, you have nothing that is useful to you for cloning right now, but now you're going to take this and you're going to run it through the second cycle. So each of those four strands, the two originals and the two newly made strands are going to be separated. You still got all those primers in there. They're going to bind in the appropriate places, you're going to extend those.
