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It. It. It. How are you doing getting over upon. It's hard to hear your professor. I know. I was halfway through my vacation. You were in the Netherlands? Yeah, that's what I got. I wonder if I went to the Netherlands. We've been there before. I had been there before and I was much. No. Museums. Museums? Yeah, museums, art museums. Talk a little bit about the course and expectations and then we'll get into some of the material. Tonight is going to be somewhat review for a lot of you. If you haven't had a course in molecular biology before as an undergrad, or if you have just been away from courses for a while, oftentimes graduate students are have been away for a little while coming back. So we're going to try to ease people into it a little bit. Speaker 1 - 06: But it is a fairly intense course. It's 5 hours a week, so it's always a little bit challenging because it's essentially two classes in one. But it does, for those of you who are in the master's program, it does give you a 700 level course which helps you for graduation. And a couple things I just wanted to mention here. So the book we're using is actually, I was hoping that they were going to come up with the 6th edition, but it looks like may or may not happen. So this is getting a little bit old. So we're going to be covering some material that is not in the book yet or that is not in the book in terms of papers.
So you'll actually be presenting one or two papers that will depend a little bit on how many we end up with here, but those will be assigned as we go through and you'll sign up for them and essentially be presenting papers on concepts that are newer or are going to integrate some of the things that I talk about here. I'm not covering the first thing chapters in the book because they are sort of basic cell biology and really basic biology stuff. So we're sort of jumping in at the stage of molecular biology a lot of stuff. Speaker 1 - 08: One of the reasons that I like this book, and I was hoping that they would be coming out with a new edition, is that it goes into much more in terms of the method to study molecular biology and not simply throwing a bunch of molecular names at you. And the same these are all the ones that are involved in this process. These are all the ones that are involved. There's plenty of that too. But I want you to understand the process by which you can address molecular questions about molecular processes, replication, regulation and transcription and translation. A lot of different methods that are used to manipulate genomes and to understand genomes, understand molecular processes. So we're going to be covering not just what is known, but also how those, how that was learned. Some of the nitty gritty details.
Like I said to some of you before, I actually haven't put anything on brightspace yet. And so I will be putting the syllabus and any updates and things like that onto it. But like I said, I just got back from vacation and I honestly haven't had any time to get into it at all. I thought it would be really easy and it's weird. So I will be putting those in. I will also be putting them in the slides so that you can see those, but I will be putting them in after I've done the lectures, not before. I found that when I put them up before people see them on their screen and tend to focus on that as opposed to listening to what I'm saying. And so I found people do better if I don't have this as a distraction. Speaker 1 - 12: I know a lot of people like to take notes on the slides themselves, but honestly, you'll do better if you listen and especially if send something out as well on how to do well in this class. But I'll post that on bright smart as well, but height space as well. But the idea is that if you read the material first so you're familiar with the concepts, then you can focus on what I'm saying and what I'm emphasizing and that will help you for studying, for example, and also learning what I'm most interested in. Okay. Okay. So I'm going to be talking about, as I said, chapter four, which is on gene cloning and molecular compilation of DNA.
But like I said, I want to get into some of the basic first things that you may have had a long time ago forgotten and things that are going to be really important understanding what we're talking about. So I'm not going to make you, for example, memorize things like structures like this, but it's important to understand what makes up, for example, a ribonucleic acid or a deoxyribonucleic acid. The differences in those, because they're going to be essential for understanding the processes that we're going to be talking about. This is a classic amp. It's got a phosphate group here on the five prime carbon here it's got the base here, in this case adenine. And the only difference between an rna molecule and a DNA base is the absence of the two prime hydrogen. Speaker 1 - 14: And that's going to be absolutely essential because of what can happen at that two prime position. But it's essential that both have a five prime phosphate group and a three prime hydroxyl group because that's where you're going to get the bond being formed between adjacent bases so that you can form these, what are called phosphodiester bonds. Okay, so you're all familiar with this, but it's going to be important as you're going to see. There are going to be enzymes that are going to be acting on RNA specifically, on DNA specifically, and in some cases both. And they'll only work on certain numbers. And the difference is so small between those molecules and yet it can have a profound effect on what's going to happen with those molecules. You're again familiar with the standard bases, AGTC and DNA.
And that is going to determine, in part, the strength of those molecular interactions. So if you have a stretch of A's and T's, that's much more susceptible to opening up those two chains, separating those two chains from one another versus those heavy and G's and C's, we'll get to that quite a lot as we go through. But you're going to have purines, the A's and G's, associated with their appropriate pyrimidines, T and C. Now, these bonds are not very strong individually, but when you've got a stretch of these holding the two strands together, they can be quite strong. And they can only be separated by either an enzymatic activity, such as a helicase, or chemically by alkaline conditions or by heat. High temperatures can also separate. That becomes really important because you can separate these, but they will re anneal. Speaker 1 - 18: They'll come together again based on their sequences, that is, the complementary sequences in the two chains will find each other again. So you can take two different molecules of DNA, double stranded DNA, and you can heat those or treat them with alkaline conditions, separate I them. And then when you return these by cooling or by neutralizing the ph, you can get them to return to finding complementary sequences. Now, this isn't perfect because you can have stretches that are complementary, that may not be the ones that they were originally associated with. So you can get all kinds of other things going on today. We'll talk more about back later on. But they will find complementary sequences without any intervention at all. By the.
By the scientific study, there are a few things that I want to point out in terms of the conventions when you're looking at DNA molecules and talking about DNA or learning basically. First of all, you have to think about two strands, which is a little bit arbitrary, but you've got a sense strand which will always show up on the top, five prime to three prime. So you'll have a hydroxide group over here, free hydroxyl group over here, often a free phosphate group here. And this five prime to three prime will, if you have the second strand, be again opposite and have the anti parallel, the opposite orientation. It's five prime here, and it's three prime here with base pairing across all of these sequences. The top, if it's the synth strand, means that the bottom is the anti stent strand. Speaker 1 - 20: Now, one thing I want to point out, and this is really important, if you're talking about a short stretch of DNA, you'll often be thinking about a synth strand and antisense strand. But when you're talking about, for example, a whole genome or even a plasmid, a piece of DNA which is relatively, comparatively small, a few thousand bases. In one case, you may have what's considered a sense strand corresponding to one gene and antisense. But just downstream or upstream of that, you may have another gene which is in the opposite orientation. So just because you have one gene corresponding to the sense strand in one position doesn't mean the entire piece of DNA is always going to be read or replicated in the same direction. See what I mean here a little bit?
We'll get to more of this later on and you'll have it down. Okay, so any questions on that so far? It's all still in your muscle memory from previous classes. Okay, so this brings me then to what I want to focus on tonight, which is going to be, in essence, a large part of historically, what's been used for what known as gene cloning, making copies, isolating genes, putting them into typically a vector, a plasmid, or a piece of DNA that is, that corresponds to a viral genome. Whatever it is that's going to vector is going to be able to hold your piece of DNA so that you can have the cells that they're in, make many more copies of your piece of DNA, where you can use it for sequencing that piece of DNA and manipulating and various other ways. Speaker 1 - 25: So gene cloning can be extremely important for many processes in. For many processes in manipulating DNA for all sorts of purposes, as well as understanding DNA that you're working with. So it allows you to manipulate DNA. You can get large amounts of a piece of DNA that you might have only had one copy of originally because you let E. Coli make more copies, etcetera, and you can get your gene of interest in a fairly pure form away from other pieces of DNA that you're not interested in. Again, you'll see why this is so important. Typically, you're going to be using DNA that may be coming from any type of organism, be it mammalian, including human or fungal or plant based or prokaryotic based.
Regardless of that, you're going to be typically taking that piece of DNA and putting it into, as I said, one of these different types of vectors. And I'll talk a little bit about these and why you might use some of these as we go through here. But what's going to be key is that in order to manipulate these DNA molecules, you need to have enough of them to work with so that you can make the modifications you're interested in. And they have to be able to grow in compatible host organisms like E. Coli or like whatever that is. So how do we do this? Speaker 1 - 26: Well, classically and for 90% of the life of molecular biology as a field restriction, endonucleases, that is, enzymes that are going to recognize a very specific sequence or small set of sequences and cut within or outside of that sequence in a particular place. Those are going to be used for taking a piece of DNA out of one context on the whole genome and putting it into another context. An isolated gene that's growing in E. Coli, these restriction endonucleases, or restriction enzymes are going to, like I said, recognize specific sequences. The restriction site make cuts in both strands, not necessarily directly across from each other. Can be a cut up here and a cut down here and I strand giving you what's called a sticky end or a cohesive end. We'll get to that in a second.
H one on the other hand is cutting over here, which means on the bottom strand where you have the same sequence, gg a, t c c, because it's in the opposite orientation, that's going to cut between the two G's opposite these two C's. That's going to give you a cohesive end, a sticky end. Okay? I am not expecting you to memorize any restriction signs. Don't worry. This is simply for you to understand that there are lots of different types you can have. Well, I'll get to this in a little bit. You can have sticky ends that are going to leave a single strand with a five prime end, others that have a three prime end, others, like I said, that are blunt. Lots of different conditions here. Some of them are going to be able to cut a number of different sites. Speaker 1 - 30: So for example, Hindi two can have either pyrimidine here, either purine here and still cut. So you'll recognize a broader number of sequences as long as it's got the GT here and the AC here. The most commonly used because they're the most practical for the ability to actually clone a piece of DNA that you're interested in and put it together with something else is to use what are known as palindromic restriction sites. That is to say, they need the same on the top strand, five prime to three prime, as they do on the bottom strand, five prime to three prime. That makes sense. So palindromic sequences are nice because like I said, every cut end is going to be compatible with every other cut end made by that enzyme.
On the other hand, if you have pyrimidine and purine here, you have to have just the right sequence for those to be able to match up. That's another piece of DNA cut with him. B two, bad example, because this is actually really a bulletin, but the idea is the same. Okay. One of the keys to understanding why you would use a four base pair recognition site or a six base pair or an eight base pair has to do with the frequency of cutting of that DNA the longer the recognition sequence. So not one, for example, requires all eight faces here. So those are going, that sequence is going to show up by random channel very infrequently compared with Alu one that only requires four cases. Speaker 1 - 32: So not one is going to cut much less frequently and create larger pieces of DNA than if you cut with Alu one. Okay. Alright. So the reason this is so important and the reason I'm going over it for you, so that you remember this, even if you've had it in other classes, is because these conditions have a lot to tell you about why you do certain types of experiments one way versus another way. Okay, so like I said, you can get different types of are generated. EcOR five reals, for example, is cutting straight through. It's a six base pair recognition site and it cuts straight through both strands in the same place. So it's going to be blunt end. And the thing about blunt ends is all blunt ends are compatible with all other blunt ends.
And that would make something cut here compatible with one of these borden cutters here. Now why is that so important? Well, if you look at the three prime order hangings, that doesn't work. The reason for that has to do with the nature of polymerases. So far, every polymerase, DNA polymerase, an rna polymerase that's been found only as nucleotides to the three prime, hydroxyl three prime, they will not add new nucleotides to a five prime atom. So they always work five different. And so this allows you, DNA polymerase is able to fill in these ends, but not fill in these ends because with a three prime overhang, it means you have a five on the other strand, five end. And polymerase can't add to that. So it's not able to do that. Instead, you can chop off the single stranded parts. Speaker 1 - 36: Again, not too important right now, but you'll see why that's important later on to make them blunt. Lots of other things that we can do with these. I'm not going to go into details right now, but the idea is that if you've got complementary sticky ends, two pieces of DNA from different sources cut with eCOR R1, under the right conditions, the right temperature, the right pH, they're able to anneal together, bind together, forming those hydrogen bonds, and then those little nicks in the backbone have to be repaired by DNA linings. Now, there are a couple things that are exceptions to what I've just told you. So anytime you have, like I said, the cohesive ends, the sticky ends that are the result of cutting with these palindromic sites is they're always going to be compatible. But in some cases, you'll have sequences.
Is there any included in this? No, there are not any included in this. There are restriction enzymes that are non palindromic. They recognize a sequence which is not the same on the top strand, five prime and B prime as on the bottom strand, five prime. And so in those cases, when they make a cut, the ends are not always going to be compatible with any other. Okay, so again, this is just a little bit of background. There are a couple of other cases where you'll have palindromic sites, but they're interrupted. So they'll have at and then nn like this one where you have gt and then you can have either one. So not every cut by Hindi two will be compatible with every other cut. Makes sense. Good. All right, excellent. So finally, in some cases, you'll have enzymes that cut asymmetrically. Speaker 1 - 39: They'll recognize the sequence, but they'll cut differently on the top strand versus on the bottom strand. And those are going to produce ends which are completely not compatible. So just to give you a better sense of this, imagine you've got this piece of DNA. EcOr five is going to make blunt cuts straight across the DNA, giving you two pieces of DNA, five prime to three prime. This was joined to this, and that's going to give your bloodhead cuts. Whereas a five prime overhang, like you get with Ecor one, that is going to give you single strands here. These are compatible with one another.
And you'll also be able to get two copies of the vector coming together and all kinds of other possibilities. But the idea behind this is you can get disparate pieces of DNA from different sources and put them together. And when you do this, when you get two half sites of a restriction site coming together, annealing and repaired, you will regenerate restriction sites. So now where you had a vector, you cut into r1 and now you put a piece of DNA again. Now you've got a new r, one site at each, one at each side of that insert in. So you can cut that piece out again if you want, using the same enzyme. Make sense? All right, so obviously there are going to be modifications to this system. Most of the restriction enzymes are from cokaryoles, so eco stands for E. Speaker 1 - 42: Coli and eco r1. R1 enzyme is capable of cutting at one particular sequence, the five reals in a different sequence. But the cool thing is that oftentimes prokaryotes want to be able to differentiate between an invading strand from, say, a virus, a bacteriophage, and prevent, and to be able to cut up that invader without bothering their own genomic DNA. And so you'll see these methylation pathways where you get methylation at an a or a c. And when you do that, methylation is going to prevent ecor one from cutting edge. That is to say, the naked DNA without methylation will be a target for that restriction enzyme. But if that site is methylated, it won't be cut by the restriction. Yes. Oh, I didn't know that, like prokaryotic DNA actually did that. I thought it was a eukaryotic thing only.
Yeah, no, this is definitely a prokaryotic thing. Yeah, yeah. But also we do carry on, but that's a different. That we'll be talking a lot more about methylation. Is that like more about like the histone? Because they don't have histones and the methylation is involved in histone modification and eukaryotes. Yeah, yeah, they do a lot of different things. We'll talk a lot more about that later on. But in this case, they're helping to use it as a defense mechanism, in essence. So what happens is these are known as restriction modification systems, where you actually get during replication, what will happen is you'll have a methylated strand, parental strand. The newly made strand is not yet methylated, but then hemimethylase comes in, recognizes that methylated sequence and does and methylates the opposite strand. So you get methylation on both of these haze in that site. Speaker 1 - 45: What's important about this and what's useful about this is we can manipulate the system by using not only the restriction enzymes that cut at those sites, but also methylases. So we can prevent cutting in places that we don't want to cut. You'll see examples of that as we go through there are a few examples, such as TPN one, and we'll talk about that later on. Those actually only cut methylated tn. And that's going to be important for different purposes. But the idea is that this system allows you to determine the relative cutting efficiency of different enzymes. So, for example, if you are working in a lab, you always want to make sure that if you are growing DNA in an E.
