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Of how they discover what lines are what and where. Anybody have anything exciting? You learned to do classes yet? Surprising or on jeopardy. That was a contributing question. Speaker 2 - 01: Oh, there was this cool thing on Jeopardy. About sneaker categories, and I learned that the Puma brand actually leaves some of their shoes after the cats. Speaker 1 - 01: Why? There you go. Speaker 2 - 01: Like, the $800. They named, like, different names of shoes. Like, for Nike, they were Air Force one. So, like, then, you know, what is Nike? Speaker 1 - 01: Right? Speaker 2 - 01: And then all star was like, converse. And then for Puma, they put, like, the different names of, like, it was like something cat. You know, it's like, oh. And then Ken Jennings was like, oh, you know, it's a cat.
You want to hear good? Jeopardy. Quick question for Canada. Final jeopardy. Landmarks of science. Poems of an original. One of these grow outside the math faculty at Cambridge University and in the president's garden at Mitz. As a matter of fact, it is. Yeah. Apple tree. Corner of the apple tree from Isaac Newton. Oh, that's. The tree is real, right? Yeah, the tree. No, that's not. Speaker 2 - 02: The tree story is real. The one where you get hit by a head. Speaker 1 - 02: Oh, I heard it wasn't. I heard that. Speaker 2 - 02: I heard it wasn't true either. Speaker 1 - 02: Yeah. Anyone else you asked? Anything interesting. That's right. To share with you. I'm afraid of talking to you because you're, like, so much above me. I'm like, what if I say something and you're like, oh, that's not interesting. Or I was like, we all knew that. Speaker 2 - 03: Oh, I made a group chat for one of my classes with doctor Denamy, and then Doctor Denamy asked if he could be in the chat, and then Delroy added him to the chat. Yeah, you can be in if you want.
I mean, it's not a very, like, you don't have any exams for this class, so, like, you know, I don't think it's very, like, you know, no one's gonna be, like, complaining after attempts, like, oh, so. Speaker 1 - 04: I have another tricky problem. I have another tricky question. Maybe I'll save that for next time. Unless you want to hear it's great. It's a chemistry tricky question. Oh, I like that. All right, quickly. Yeah. This element was first isolated in 1945 during uranium fission research. This element was named after the ancient deities suggested people getting a new power. Don't you need people getting a new power? Yeah, I don't know why I can't do something for me. This is March 5. Speaker 2 - 04: Oh, it was final jeopardy. Oh, that's always a hard question. Speaker 1 - 04: So that's the fun part. I think it's interesting that maybe story. No, that is a real thing. Atomic number six. That's the breathing fire. Yeah. Because it brought fire to you. Yeah. Speaker 2 - 05: See, when you said 1945, I could think about the atom bomb.
Yeah. All right. It. All right. So tonight will be the last of the sort of rapid movement through a bunch of techniques, and you'll see why these are so important reason that I'm going through all of these starting next week when we start talking about transcriptions and in fact, how we know about the interactions between things like rna polymerase with DNA and prokaryotes. Typically, I start off with discussion of prokaryotes. They tend to be, for most processes, simpler than the corresponding method in eukaryotes. And so with the prokaryotes, we'll be discussing things, and I'll be then referring to those and the modifications, differences between the eukaryotic and the prokaryotic systems after that. And that's going to be really important because you can see why it is. Speaker 1 - 07: For example, last time I talked about how, if you're putting a eukaryotic gene into a prokaryote for expression, you have to not just put the DNA sequence in, but it has to be compatible with the systems for transcriptional translation in the prokaryote. And typical, the typical elements in eukaryotes will not work in prokaryotes. They have different sets of proteins, they have different recognition mechanisms, both for transcription and translation, and everything in between. So you'll see there a lot of similarities, but enough differences that there are a lot of things that you're going to have to learn the differences in why things work in one way in a prokaryote versus in a eukaryote. You'll see what I'm talking about as we go.
In order to understand how things work, we need to understand the DNA. And we talked about this before about how DNA is the starting point, of course, when we're talking about gene expression, we've got the DNA, we've got the modifications to the DNA regulation of transcription off that DNA, taking the transcript and translating it. All of these different components are dependent on sequences. The makeup of the DNA, the rna that comes from it, where that rna begins, how that RNA is regulated in terms of its polymerization off of the DNA template sequences of the proteins, individual amino acids. And understanding that will really help you to, again, think of these concepts at the most basic level. Speaker 1 - 11: Okay, so I talked at the end of Tuesday's lecture about southern blotting, and southern blotting, as you know, specifically, southern blotting is DNA transfer from a gel to a membrane. That membrane can then be probed with a nucleic acid that will recognize complementary sequences and be able to identify, based on whatever is associated with that probe. We'll be able to identify particular sequences from among millions or thousands of bands on a gel that you can't see from one that you can see because the probe is magnitude. Okay. So there are a lot of things that we can do using those methods, and you'll see these over and over again throughout the semester.
But understanding sequencing and some of the ways that can be used for probing DNA, as well as identification of things, many different fields, we're going to use southern blotting that allow us to, for example, identify a particular individual or a particular mutation, a particular modification, whatever it is, from, in this case, genomic DNA. And we've talked about this before. One of the things that we can do, especially with eukaryotic systems, is to find markers that are going to be variable from one individual to another. In some cases, we want to know differences between my DNA and Robert's DNA. Others are going to want to be able to differentiate between human DNA and chimpanzee DNA and apple tree DNA. Speaker 1 - 13: Okay, so one of these mechanisms is the use of microsatellites and mini satellites of DNA, which are simple, but highly variable components that are found throughout the genomes that are represented as a result of the way replication occurs. And we'll be talking about homologous recombination later on when we talk about replication. But understand that there are going to be short sequences of bases, sometimes just three bases long, sometimes six bases long, whatever it is that is repeated. And that repeat sequence can represent 20 copies, of that repeat, 50 copies, ten copies, and those can change even from one individual to their offspring. So you can get these sequences which allow for DNA fingerprinting to say, in the case of forensic analysis, this is the individual that corresponds with this piece of DNA in a particular crime scene or an identification, something like that.
This sequence will be the same for this individual. But the number of copies of the repeat will be different. And so the size of that fragment, this one will be bigger because it has more copies of that repeat. Okay, so when you look at multiple loci of these microsatellites, of these markers, these are variable enough that on the order of one in 10 billion, there's a one in 10 billion chance of having two individuals, even related individuals, with the exception of twins, which can be quite identical, that there's one in them, 10 billion chance of finding the same pattern between two individuals. So they're highly selective. And we're talking about this not just in terms of forensics, but we're talking about it in terms of parental identification, of cases of mapping particular genes within a genome, etcetera. But here's the idea, okay? Speaker 1 - 18: So let's say you've got these sites here, different microsatellites with different numbers of copies of that repeat. We cut with ecor one in this case, or, no, a three. Excuse me. You cut those and you get a pattern of bands. Can't tell anything about the bands yet because there are a few bands among all these others that you can't see yet. But when you blocked now onto a membrane and then you hybridize with that microsatellite DNA, you identify those three bands of those particular sizes. Once you have that, now you can compare this individual set or pattern of bandst that all have that microsatellite sequence. But the sizes of those can be quite different. So this is an example of that in this particular case. So we have these nine different individuals here, as well as these two, which are monozygotic twins, identical twins.
And you can see that each one of these has a different patterns. Some of the bands are the same on several individuals. But when you look at the entire pattern overall, no two individuals here have the same pattern, whereas the monozygotic twins will have the same pattern because they came from the same original zygote. So, like I said, you can use this forensics, but it's useful for a lot of other things as well. Where you can, in this particular example from the book, they've got the marker lane, so you know what the different sizes are. So you can compare them, put them in a database somewhere that we didn't ask for. You've got different suspects here. A sample of semen which has this same pattern as suspect bhdeminal. And then a vaginal swab also shows that, whereas. Speaker 1 - 20: And that's not the victim's DNA that's down here, different sized bands, random control DNA. And so what you can say based on this and with three markers, that wouldn't be sufficient, but that it is likely that suspect P is responsible for that seen in sandbox. Okay, so that's the idea behind this. But like I said, this has a lot more purposes than simply location of suspects in a forensic setting. And there's a lot more to this than just three individual markers in the DNA that would not be sufficient to. To identify that particular individual at all. But when you're talking about 20 individual bands, you're talking about an exponential likelihood. The more of those you have that if they match, that you have narrowed in on particular individual.
And they're comparable because in the case of, for example, animals, there are some sequences that are highly similar in mitochondria, for example, that allow you to identify regions, make primers to two regions flanking this variable region, and say, yes, this corresponds to this particular organism. Okay, so this ends up being really powerful. I'll be talking a little bit, not today, but later on about edna, for example, environmental DNA, which is basically tiny amounts of DNA that can be amplified from where an insect has been walking, which sounds crazy, but in fact, Doctor Coleman does some of these experiments and I'm helping her with some of them, looking at skunk cabbage and what's been there to warm up in the wintertime because they're thermogenic and ten degrees warmer than the outside temperature. Speaker 1 - 25: And so you get these insects going in there that can end up pollinating the flowers, but at the same time also warming themselves up. So it contrasts these. So it's kind of cool. But that's the idea behind some of these. But the DNA barcoding is much more broadly used. The idea is that you're going to take a DNA, or in some cases an RNA sample, you're going to generate primers that are going to recognize, again, PCR primers that are going to recognize the region flanking these variable sequences. Then you make the PCR product, you sequence it. We'll talk about how to sequence here in a little bit.
But you sequence that DNA or rna, you compare it with those sequences in the database and say, this particular gene that we're using, as our standard turns out, to show that we're dealing with a particular virus or a particular mammalian species or a plant that we didn't recognize because it wasn't flowering at the time. Lots of different things that you can do. So typically, for example, in animals, they'll use cytochrome c oxidase, which is a mitochondrial gene, works really well with animals. Even though plants have it doesn't work as well for them because they're much less variable. And so you don't get that variability that allows you to identify based on the sequences. You can use that information to identify a group and in some cases a lineage, mitochondrial or a female lineage. In some cases in plants, oftentimes they'll use a chloroplast gene. Speaker 1 - 27: The large subunit of Lubisco, or the mature k gene, that, again, allows them to do the same thing. It's got conserved regions at the end and variable regions in between. Others will use variable regions of ribosomal rna's 18s fungi, some eukaryotic microbes, or the 16s ribosome rna. You'll learn about these later on that are from prokaryotes and allow for identification of those. And so this provides an enormous advantage, speeds up identification of what you're coming across, whether it's in a clinical setting or whether it's in an conservation biology setting, to be able to identify these. And in fact, they did this not. Not too long ago, they went to fish market here in Flushing, and they actually took samples of different fish that they were told, this is herring, this is Tod, whatever.
But one way to identify a particular sequence, especially if you have a repeat sequence like a microsatellite, you can actually find those sequences in the genome directly, visually them, by taking chromosomes. And in some cases, you can do what's called combing chromosomes. Essentially, you're taking chunks of chromosome and you're stretching them out. So you have single copies of the chromosomes, and then you can probe those with tags that are going to allow for binding to known sequences, recognized sequences with certain patterns that show up in the genome. And then you also probe with your sequence for your gene of interest, and you can see where your gene is relative to these other points within the genome. Speaker 1 - 31: Prior to things like this, the only way of locating a particular gene relative to other genes in the gene was to do mapping studies which can take weeks, months, years in some cases, whereas this you can do in an afternoon. It can be very fast. That being said, even this is becoming less and less common, because especially for genomes that have been studied extensively and are sequenced, it becomes possible to simply either look at a database. If you're trying to find a particular sequence, you don't need to go through all of this process, or if it's something that is fully sequenced but hasn't been fully annotated, hasn't been analyzed particularly. You can in fact take your sequence and you can say these are likely components within that genome, where this is coming from. Speaker 1 - 32: This is likely an ATP binding protein, this is likely a sodium channel, whatever it is, based on the sequence. And you can locate those, again in silica, that is looking at the sequences on a computer. All right, so in order to make this work and this is going to be common again, you're going to see these same basic processes going on over and over again in this pipeline.
In this case, you're going to be combing those chromosomes, like I said, so they're not all tangled, but you have this little linear stretch here, and then you have to partially denature it so that you've got single stranded regions that are going to allow for a probe to bind, and then you stain the chromosomes, or if they're fluorescent, you can identify them directly and identify, based on the pattern which chromosome you're dealing with and where within that chromosome, relative to the telomeres and the centromeres, for example. Speaker 1 - 33: And then as signposts within the genome, you can use known probes that bind to known sequences that are going to say, this is toward the north arm of chromosome three and about two thirds of the way, and then you can start looking for known posts around that region so you can get a much better sense for where your gene of interest is. So if it's with a fluorescent tag you're dealing with, this is known as in situ hybridization, fluorescence in c two hydrolysation, which actually is a method that corresponds to a lot of things. In this case, it's dealing with direct labeling of DNA. But you see in situ hybridization, the fluorescent probes for all kinds of purposes. Some of those we'll talk about later. Anybody still with me?
But you're going to spike, you can have four different tubes and you're going to spike each one with one of the nucleotides that is missing that critical three prime hydroxyl group. So once they incorporate a dideoxynucleotide into that growing strand of DNA, all of a sudden you put in one of these and you can't add any more nucleotides because you need that three prime hydroxyl group to add the next two. So what that means is that if you're starting from the same place and you're going up the sequence and you've got a bunch of copies of template that's all the same, it means that as it's building this new chain of sequences, in some cases, the first a you get to it will stop. Another instance in the same tube. Speaker 1 - 37: It may not stop until the fifth a because it would have put in a normal deoxy nucleotide, and then it puts in a dideoxy and that stops it. So you get this whole bunch of bands of products, all of which end in an a. In that first two. The next two, you have dideoxyt instead. And so that's going to, again, stop with all the t's and then the c and G. So all four of those are represented. And what you get is a, when you separate these on a gel, basically, based on the size of these and where the band is present in the a column or the t column or the c or the g, that will tell you what goes there. So the idea is that.
So this is known as chain termination sequencing, Sanger chain termination, because it's making a new DNA strand, a new DNA chain, and it's terminating because of that dideoxy. So it looks like if you imagine in this particular case, you've got a primer that you're adding, and you begin polymerase begins adding nucleotides, and in this particular case, the second t that it gets to, it puts in a dideoxy and it stops making the rest of the strand. So this will be a very short band corresponding to just the primer plus these few nucleotides. So it will be way down here toward the bottom of the gel, because those smaller bands go fastest. For each one of the four tubes, this one has dideoxy a, dideoxy c, g and g. Speaker 1 - 39: You get all of these different products corresponding to things that are ending or trying to incorporate an a and put in a dideoxy a, trying to incorporate a c, but put in a dideoxy and it stops. If your template is good, all of them the same, then it means that you're never going to have two bands that are the same size that end in different things, right? Because they're starting in the same place, they're continuing until each one of those. That means that you can tell what that last, where that last c was or that last a was based on how far it migrated down the gym. So in this case, you've got a c, g and t. So you're going to have here a c being closest to the primer.
