 Thanks very much, and everybody hear me all right. Yep, okay, good. So I'm Bob Wilden. I've been here at NHGRI about nine months. I'm an MD clinical geneticist with a background in research and software development, and I came here to help NHGRI do whatever is needed to bring genomic healthcare into general medical practice. And education is one of those things, and so this is really actually very important to me and to us at the Genomic Healthcare Branch, and I want to thank all the people who've really helped put this together and invited me to do this, and I want to thank you guys for being here because you're really important in all of this as well. So without further ado, which button here, okay. So the general plan here is to first talk about DNA, that's an acronym, we're in the government so we have to talk in acronyms, and this is, and then genome, and then we're going to talk about replication and variation. We're not going to talk about gene right away because I think all this is actually really important to understand all of this part down here. We have a two-hour session here. I think we're going to take a break in between the first hour and the second hour, and I'm not sure exactly where the break is going to fall, but maybe right in here. And then we're going to talk about some more complex and human-oriented issues in the second half. So that's the general plan. Okay, so what about DNA? And let me say first, please interrupt me, raise your hand if there are questions, if I'm going too fast, if I've said something that isn't clear, if I see you guys looking at each other, and please stop me. This should be a little bit interactive or a small enough group we can do that. Okay, so DNA is where? So it's in every cell, and a cell is kind of like an encapsulated system. So this is a diagram of a cell that's been cut open, and inside this little styrofoam looking ball is the nucleus, and it's been kind of cut open, and most of the DNA in the cell is inside the nucleus, and is packaged as chromosomes. So that's the most commonly thought of place of where the genetic material resides, but there's another hidden place. Anybody want to venture where that is? mitochondria, okay, so the mitochondria over here is more than one mitochondria per cell, the mitochondria inherited from mother, not from father, which makes the inheritance interesting. And so that's pretty much what we're talking about. So what is DNA? Anybody recognize this figure up here? Okay, so that's the X-ray crystallography picture of DNA. And that was critical in identifying the structure of DNA. So DNA is a chemical, it's deoxyribonucleic acid, and we're gonna go into more detail on that. It has a structure, which is this double helix, and it contains information. And this is my little panel to show that a string of any kind of letters doesn't necessarily have information. It's the order of those letters that creates information. And so the sequence is the order of bases in the DNA strand. And the genetic code is the way the information is interpreted for protein sequence. But there's lots more information in the genome, we're just not sure how to interpret it, okay? Because that's not based on this genetic code. All right, so the basic structure is two strands of the sugar phosphate backbone. And we're gonna get a little more geeky about this in a few minutes. And the base pairs are bases that are attached covalently to each strand. And then attached to each other via hydrogen bonds. And those are called base pairs, all right? So I put here how to get it. And I think you're gonna do this or watch the full video tomorrow morning. But I just wanted to give you a quickie here. Hi, I'm Dr. Eric Green. I'm the director of the National Human Genome Research Institute at the National Institutes of Health. And I'm here with my wonderful sidekick. I'm Dr. Karlie Easter also at the National Human Genome Research Institute at the National Institutes of Health. And I'm the education specialist. And today we're here to isolate strawberry DNA using household things, I guess. Basically, we are going to show you how to get DNA out of a strawberry. Great, and why do we pick strawberries? Well, so for me, there are many reasons. The first of all, they smell really nice. They're very pretty. The other thing is they're nice and edible. And their seeds are on the outside of the strawberry. And I have found that it's very easy to get DNA out of a strawberry. Yeah, although, of course, like all living things, they have DNA. We could pick a variety of different other things to purify DNA from. We just pick strawberries because it's easy and they're easy to get. There we go. So OK, you ready to start? Yep. OK, so you're going to do more of that tomorrow. So let me go back to this. Any questions about where DNA is? Even in strawberries. OK, so and how fragile is DNA? Anybody have any ideas how fragile it is? No. Can you pick it up off the microphone in front of you been there for a while? Yeah, so DNA is not fragile. It's actually very durable stuff. You can split the easiest way to disrupt it is split down the middle, split the base pairs down the middle and make two strands. You just do that by heating it up. But if you let it cool down slowly, it'll go back together. So that really doesn't destroy it. You can tear it, shear it like that, and cut across this way. And you can break it into smaller pieces, but you're not destroying the DNA. And you're only destroying the information in the case of ripping a page in half of a book. You can still read most of it. So and it can stay around, actually, for thousands of years. So it's actually really durable stuff. And that's something that I think most people don't realize. I'm going to talk about some confusing points about the DNA structure, things that I think people in general get confused about, and particularly students who are learning it for the first time. One is this sort of double helix. So the double helix is the structure. And that's what was generated by this X-ray crystallograph. And it has these two strands that are helix around helix. So they're just kind of curving around each other. So I think of the double helix as the structure. And then the strands have opposite direction, which we'll go into more in the next slide. The double strands, this is double-stranded DNA versus single-stranded. If you split it down the middle by heating it like I talked about, then you get two single-stranded molecules. And there's confusion between the double strands and having two gene copies versus one gene copy. So this single molecule of double-stranded DNA will be one copy of a gene. And in diploid organisms like we are in eukaryotes, you have two copies of almost every gene. So you actually have two of these. And that's the two gene copies that we talk about when we talk about the copy we inherit from mom and the copy we inherit from dad. So those are concepts that double-stranded is the structure of the molecule itself. And then the two copies is because we have two copies of these. Great, so now we're going to get a little bit geeky and get into chemistry. So double-stranded DNA-based pairing. It's deoxyribonucleic acid. So if you cover up the deoxy and say, well, let's try to break this down. So it's ribo, which is about ribose. It's this sugar molecule here in the sugar phosphate backbone. That's the blue things that form the backbone of the helix. And nucleic means it's found in the nucleus. It's sort of named after where it was found. And then acid, it's actually a mild acid. And you can tell that if you've ever seen DNA fragments separated on an electrophoretic gel, it moves toward the positive, if I'm getting this right. Yeah, it moves toward the positive terminal because it's negatively charged like an acid that's been ionized. So it's really a chemical. It's a complex chemical, but it's a very predictable chemical. So it has these sugar molecules that are connected with phosphate molecules. And there's a little bit more detail showing in a minute. And then attached to the sugar molecules, sort of pointing into the middle of this helix between the backbones are the bases. And there are a number of different ways in which we diagram the bases. And this is one sort of cartoonish way to diagram the bases that demonstrates by the means of these sort of lock and key fit that adenine tends to pair with thymine and guanine with cytosine and so forth. And that these two strands are really held together only by the hydrogen bonds between the bases. The other thing before I move on to that is to note that these two strands are actually moving in opposite directions. They're not moving, I should say. They have an orientation. So these sugar molecules, if you look at this point, it's pointing in this direction and the sugar molecules pointing this direction. So they're actually really identical chemicals. So if you take this side and turn it over the other way, it'll be pointing the other way just like that. That's different than, say, having one of those hooks that you hook your garden gate with that has a hole and a hook. Those hooked together, they're complementary, but they're really not identical kinds of things. And that's one of the really cool things about DNA is they're complementary, but at the same time, identical if you ignore the base pairing for the time being. So the other cool thing about DNA is it has these bases that base pair with each other. And the base pairing is by hydrogen bonds. And what I think is really cool about this is that hydrogen bonds, hydrogen, if you get into real chemistry, is a univalent molecule, which most people think, OK, hydrogen is univalent. That means it can only form one bond, right? But hydrogen, it does this, really needs something called, it has a covalent bond, it forms there, but it also can form these weak bonds, these hydrogen bonds, with a particular oxygen or nitrogen. And the cytosine and guanine pair have three opportunities for hydrogen bonding, whereas the thiamine and adenine have two opportunities for hydrogen bonding. And what that means is that this pair actually binds tighter than this pair. So if you have a stretch of A's and T's in a row, that's going to be easier to pull the two strands apart than if you have a stretch of C's and G's. Just something to keep in the back of your mind for maybe some of the stuff that's coming up later in the course. All right, seems to me there was something else I was going to talk about. Any questions about this? What is a genome? So is this a genome? No, this is a wallaby. I put this up here because we don't really have a good way to diagram what a genome is, right? There's no sort of picture of a genome. And in fact, I'm trying to find one. This is the closest I found. So this is an electron micrograph of a genome of a bacteriophage. Anybody knows what a bacteriophage is? So it's a virus, tiny, tiny, tiny, tiny virus that infects bacteria. That's how tiny it is. And it has one DNA strand. And it has just enough information to tell the bacteria, make more of me, OK? So that's a very, very simple type of genome. So the human genome, on the other hand, is really in a lot of ways the same. It's a huge amount of DNA. It's a lot more DNA. But it's saying how to make more of me, OK? And it's made of DNA, which we've gone through. It's all the genetic material in the nucleus plus the mitochondrial genome. It has molecules of DNA that contain the coded instructions for how to build, maintain, and replicate a human being. OK, now I have an interview here I want to show you about how kids view genes. Ben Thomas came to the museum with his family from Dayton, Ohio. Are you able to see any more of your DNA? The 12-year-old is ready to embark on a genomic journey. I have always found it really intriguing that everything about who we are and what we look like is controlled by these tiny molecules called DNA. And this exhibit is just like cake to me. OK, so if you're in high school, it may not be cake. It may be sex or something else. But it's really cool, OK? So let me get out of this. And that video was taken at the Unlocking Life's Code Genome exhibit, and I don't know whether you've already been told about that. It was an exhibit at the Smithsonian, and now it's traveling around the country, and it was developed by our folks here, and along with the Smithsonian. And it's in St. Louis now, and next it's going to Portland, Oregon. So if you're in those areas, please keep an eye out for it. But I thought that was a really neat way of a kid really getting what a genome is, OK? Human genomes are not identical in anyone but twins, OK? They're very close to each other, but they're not identical. DNA, the genome always contains both benign variation, variation that doesn't cause any problem from a medical or life standpoint, and variation that can cause or contribute to diseases, OK? So all of us carry genetic changes that in the right circumstance might cause disease. It may mean that we have to have two copies of those, that particular variant, to cause disease, and we only have one copy, so we're a carrier. We'll talk about that more later. But we all carry those kinds of things, OK? So nobody is completely clean, so to speak. And the human genome is really big. It's 3.3 billion base pairs, which is a lot of zeros, and it's actually twice that because we have two copies, right? We have a copy inherited from mom and a copy we inherited from dad, OK? Lots and lots of sequence. All right, the human genome and eukaryotic genomes, well, all genomes, in fact, are organized into what are called chromosomes. So a chromosome is basically one strand of DNA. It can be really, really, really long, or it could be short, OK? And a bacterial chromosome is a circle, OK? Viral chromosomes tend to be single, short, linear molecules. And human chromosomes, like other eukaryotic chromosomes, are these really long pieces of DNA that are packed together. So we're trying to pack a lot of information into a small package. We start from the sort of the DNA strand, and we add histones, and then those stick together to form nucleosomes, and then we wind those nucleosomes up, and then we wind the wound up nucleosomes into tighter and tighter packages until we get something, at least during cell division in prophase that looks like a chromosome that we can see under the microscope, OK? And just to take this opportunity to show the parts of the chromosome, so this is a replicated chromosome. It has two sister chromatids, and the ends are telomere, it's for you, linguists, sort of telomere like telephone, far away, and centromere, central, and then the P-arm and the Q-arm, which is the short arm and the long arm. And the only way I can remember the difference that's hard to remember is to think of P as petite, which is French for short, all right? That's how I remember it. And then the chromosomes themselves are packed into the nucleus there. So when we do a chromosome study in medicine or in the laboratory, we get a picture that looks like this. We put the cells through cell division, and we catch them at just the right time when the chromosomes are really condensed like this. And then we take a picture of it under the microscope, and then we used to actually literally print the picture on paper, and then we would take scissors and cut out the individual chromosomes and stick them onto a piece of paper that had these numbers on them so we would array them. So there are 23 pairs of chromosomes, and they're basically organized by the length, OK? So before we had this ability to barcode them, sort of stripe them, they just look like pieces, and we would order them by their length, OK? So that's where the chromosome numbering system comes from. And now we have a ability to, well, for many years now, to add a stain, which shows different stripy areas, and that allows us to sort of see which chromosomes that are almost the same size, how they're different, and which ones are actually which, OK? So 22 pairs of autosomes in the yellow box and one pair of sex chromosomes in boys, that's an X and a Y, and girls, it's two Xs, OK? This is probably not new to you at all. We're just reviewing a lot of stuff. They're packages of DNA, and they have a consistent structure within a particular species, OK? So I threw, this next slide is not in your print, but this is a comparison between human, chimp, gorilla, and orangutan species, and it's the same chromosome sets, but in diagram form, OK? And what you see is that a lot of places, the banding form, the banding system is entirely the same. And in other places, there are little differences here, and then sometimes the chromosomes are broken into two parts, so here's, you know, here and here are two different chromosomes that represent the human chromosome number two in the other apes, OK? So a lot of it's the same material, but it's kind of organized a little bit differently. All right. Human genome project, who's heard of that? Yay, OK, does it come up in your classes a lot? Does it come up a lot? OK, so I'm not going to talk a lot about it, but basically to say that the human genome project was this really big deal that came out of NHGRI, and the concept was that we're going to sequence the entire genome. We're going to get the DNA information from the entire genome, starting with the genes, really. And we're going to, that's going to push us forward in terms of knowledge and ability. And I will tell you that as a scientist who was trying to get grant support for simple, direct, basic disease research at the time, I was not very happy about this because it cost a lot of money to do this. And it was brand new technology, and it was risky. And what they were doing was saying, we believe that after we get all this information, it's actually going to be useful. But they hadn't proven that. And if I were applying for a grant at the time and said, oh, I'm going to do this big thing, and afterwards I'm going to have all this information, and it's probably going to be useful, it would have gotten rejected. But these guys were actually really smart. And I'm really glad they did it, even though I was not very happy at the time. So because it really has panned out to be a really tremendous boost to not only genome research, but also research for all kinds of biology and human diseases. So it's become really this amazing tool rather than a result in itself. It's become a terrific tool. So here's kind of the timeline. And then this human reference sequence was complete. And we really were excited about that. And over time, we realized that, yes, that was really great. But yes, they're actually holes. And yes, this piece doesn't actually connect with this piece. So this project actually is sort of continuing in some ways and that it is refining the information and the quality and the information that was gathered at that time. OK. This is sort of my timeline description of what was actually done. So the first thing was done was not to sequence, because we didn't have the technology to sequence the whole genome. The first thing was done was to map the genome. And the reason I raised this is because people confuse these two things. And I think that's helpful to distinguish sequencing the genome from mapping the genome. And mapping the genome was an early part of the project where through sort of traditional genetic techniques applied in very high throughput technology systems, we were able to put sort of markers all along the genome, sort of mile markers. And so we were able to identify kind of where the towns were and where the major population centers were. But we didn't have the information about every spot on the road in between. OK. And we were able to tell which towns linked up with which towns. And then we began to get more detail by sequencing and began to get this idea that, yes, there were houses there, there were trees, there were other things in between by sequencing that we were able to get to. And then we were able to get down to the point of seeing even sort of the shingles on the house. OK. So let's call the shingles on the house the base pairs and the house would be a gene. So that's kind of a metaphor for how the genome project progressed. So that generated the output of the genome project was huge amounts of sequence data. Sequence data we store in text files, basically, where we put down the base sequence, ACGT. This is a very simple file format called FASTA, F-A-S-T-A. And this is the sequence of one gene called FoxB3, which is one I used to work on years ago. And what this slide doesn't show you is there are about six more screens full below this one just to describe this one actually quite small gene. So when you're talking about 20 plus 1,000 genes and many of them most of them are going to be bigger than this. And when you're talking about the gene part of the genome being just 1% of the genome, you're talking about a lot of sequence. So we're talking about information technology being absolutely critical for this process. All right. DNA replication. All right. So DNA replication is important because for a lot of different reasons, but let's basically go through it. So we have a double-stranded DNA. And we want to make two copies of it. We want to make one strand, one copy of the double-stranded DNA into two copies of double-stranded DNA. And that's important because we need to do that before we can duplicate cells, before we can make two cells out of one cell, before we can make two organisms out of one organism, for example. So the process is first that you have to unwind the DNA. And then you have to take an enzyme called DNA polymerase. So DNA is a polymer, right? It's a bunch of sugar phosphate bath bones and bases. It's a polymer. And ace, that suffix ace, generally means it's an enzyme. So DNA polymerase is an enzyme which makes a polymer called DNA. And it does that by attaching to a single-stranded DNA with a little short sequence that's attached to it. And it sees that open thing and all these open bases as an opportunity to add more bases. So if you have the DNA polymerase, you have the single-stranded DNA, and you have a starting point, then all you have to add is nucleotides. And the DNA polymerase will say, OK, ready? I'm ready to copy and I copy. So you need DNA polymerase. You need nucleotides. You need template. You need ATP. What's the ATP for? Anybody know? Energy. It provides the phosphate and the sugar phosphate backbone plus the energy to make those bonds form. It's a directional process. It goes from 5 prime to 3 prime. And the information is preserved, because a base will only be added if it's complementary to the base on the other strand that's being copied. So it's complementary, but it's the same information. You can copy it again on the others. Copy your new strand back to the other strand, and you have exactly the same thing. All right. Questions about that? Yeah. This may be very low level, but I get confused about when is this happening in a cell? Is it at development or throughout life? Maybe it's very simplistic. No, I think that's a great question. So the question is, when does DNA replication happen? And it basically happens anytime you need to divide the cell. So if a cell is just sitting there, like most of our brain cells, they're not dividing. They're sitting there doing stuff, but they're not dividing. So whenever you need to make another cell out of two daughter cells out of one cell, you need to replicate the DNA, because the cells need those instructions. We're going to go through later mitosis, which is that cell division process. And then even later, we're going to go through mitosis, which is the process that makes germ cells like sperm and egg. So this is a video of, I think I have to click to make it go. It's an animation, but I think it's using computer animation based on molecular research. We are now able to see how DNA is actually copied in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two cells. It unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand, because it must be copied backwards. It is drawn out repeatedly in loops and copied one section at a time. The end result is two new DNA molecules. So I think that's kind of mesmerizing in addition to being informative. What they were talking about was that when you're unwinding the DNA in one direction, remember the strands are going opposite to each other in terms of their orientation. So one of them can be copied five prime to three prime directly, and the other one, you have to go a little ways down and copy backwards because the polymerase only goes in one direction. So you have the replication fork and the leading and lagging strands. So those are the terms that are used, I think you'll hear about. And I thought the video was kind of helpful in showing that. All right, how do we use DNA replication? I think this is really, really important as well. So we use it for technology, right? I mean, it's been completely co-opted not just to divide cells, but to do all kinds of stuff in the lab in the test tube. One of them is most DNA sequencing technologies are based on DNA replication. And this background here is not the latest DNA sequencing technology, one that's probably 15 years old, which in all these different colored squiggle lines are the lines that represent the positions of A, C, Gs, and Ts, each one with a different color. And basically, you're sequencing down the way and stopping every time you reach an A, every time you reach an A, every time you reach an A, and that's what these peaks represent. And you have another tube where it stops only when you reach a C, a C, a C, a C, and that's what the C peaks represent, the blue ones. So there are lots of different ways that you can use the sequencing technology. Who's heard of PCR, polymerized chain reaction? Wow, that's impressive. PCR is a technique which was developed in the mid-1980s, basically, to take an input DNA. And if you know the sequence, you can design starting points for it, and then you exponentially replicate the DNA in a test tube. And it's a really cool process, and it was another one of those real kick starters in terms of the technology that allowed us to do a lot of stuff, including the sequencing that was done in the human genome project. Mutation detection techniques, some techniques that use the polymerase to go down the line until they stop because they're missing a particular base. DNA diagnostics, a lot of them are based on replication. And then things like reconstructing agent genomes, where you only get a little tiny fragments, but the fragments may overlap. And you can use polymerase to pull them together. I'm going to go back here and just say, so this DNA polymerase plus nucleotides plus template, you can do in a test tube. You can do a tiny little test tube. It's actually really easy. And one of the cool things is that you use a DNA polymerase from bacteria. Bacteria make it, you extract it from bacteria, you put it in test tube with human DNA. Does it matter? No, it doesn't matter. DNA is DNA. It's a chemical. The information in the DNA may encode human genes as opposed to bacterial genes, but the polymerase doesn't know that. It's got a job to do, so replicate it. That's it. I think that's pretty cool. Replication versus mitosis. So this is another area where people can get bogged down because we're talking about making two of one thing in both cases. So replication is the process of making a copy of DNA. And we've just been talking a lot about that. Mitosis is the process of replicating the genome and separating the two copies in cell division. So here's a diagram of mitosis. And we're starting out with one diploid cell, so one cell with two copies of both genomes, mom's genome and dad's genome. And the DNA is all spread out. It's not condensed, and that's called chromatin. You have 46 chromosomes, two sets of 23, or two in. And then what you get out at the other end is two diploid cells with 46 chromosomes each, each with two in. So you're taking one cell, you make two cells. That's mitosis. The steps are to condense the DNA into chromosomes, replicate the DNA. You have a sister chromatin. Now you have two sister chromatins on each set of chromosomes. You organize them. You line them up in the middle, and you pull them apart into two sets. And somehow they know that only one of each chromosome goes into each daughter cell. And then in telophase, you're separating the cells, and you get your final cells there. So one other point that often is confusing for students, I think. So this is my diagnosis. Mitosis is what you want your money to do, right? Just you take one, and you get two. So centrosome and centromere. So these are sort of anatomy of the cell kinds of things. The centromere is that place in the middle of the chromosome that's constricted, and where the two sister chromatins stick together until anaphase when they're pulled apart. The centrosome is this little microtubule organizing guy that's out in the cell. And it replicates as well. So you get two during mitosis. And it basically forms the organizing point for the microtubules that go out and grab the centromeres from each sister chromatid and pull them to opposite ends. So centrosome and centromere, OK? DNA replication is not perfect, OK? DNA replication machinery has a proofreading function. And sometimes it's built into the polymerase itself, and sometimes it's an associated protein. And the idea is that it doesn't want to make mistakes, because if you garble everything, then you don't have instructions anymore. You have garbage, right? So you want to be pretty darn good at replicating the DNA because it's important information, OK? It's like sending your email and having it come with every third letter messed up. It loses the information, and that's not good. But nobody's perfect, OK? It's not perfect. The replication isn't perfect. And sometimes that may not be a bad thing. So I want to stop for a second and have you pair up with whoever's near you and talk about why, just for two minutes, talk about why it might be important for replication not to be perfect. OK, wrapping it up. Did we figure it out? At the far end of the table, you want to start? No? Anybody? OK. Yeah? Does anybody want to put dot the i's and cross the t's on that idea? OK, great. So if you think about it, if you think about that original DNA molecule back at the origins of life, what would happen if it were perfectly replicated all the time? You would never have variation on which to select for the environment, for advantages in the environment to improve. So basically, the fact that the proofreading isn't perfect is what makes evolution run. OK? And sometimes this is hard to explain to people, but this is the way I explain it. So why do people get mutations that cause breast cancer? Well, replication errors are part of life and they're part of life in part because without them, you don't get this process of evolution. OK? Any questions about that? OK, great. You guys are really smart. OK. So there are other things other than proofreading errors that cause DNA to not be completely have total fidelity in living and life. So there are mutagens, like chemicals. And I put this thing up. I found this on the internet. I thought it was kind of interesting. So in California, you always see the labels that say, contains carcinogens. And then you eat off it. So ultraviolet light is something that DNA is very sensitive to, OK? The bases in the DNA structure, getting back to physical chemistry, actually absorb UV light. And in the laboratory, that's how we measure how much DNA is in solution in our test tube. We put it through a spectrophotometer that shines UV light on it and says, well, this much UV light was absorbed. And we then calculate from that how much DNA is in that tube. If you have a lot of UV light, UV light will damage the DNA because that energy is absorbed and eventually it breaks the bots. Now, there are in organisms, specific machinery that goes around and looks for the type of damage that UV light produces and prepares it. It's not perfect either. That's why I have age bots, all right? So we have UV light damage for DNA. Ionizing radiation, that's like x-rays. If you get too many x-rays, you can damage your DNA. And then the things that are more complex, like DNA repeat instability. So there are segments of complex genomes, like human genomes, that are actually repeated. So there are 2,000 bases that are kind of in order. And then they're repeated again next to that. And they're repeated again next to that. And they can be repeated hundreds of times. But those repeats can become unstable or they can form loops during the replication process. Because if they're repeated, during the replication process, those 2 strands can get intertwined. And you're not sure whether you're the part down here or the part down there. And you can actually delete or duplicate big segments of DNA. There's a category of human disease called triplet repeat diseases. And those are just CGG, CGG, CGG, for example. And those triplet repeats in certain contexts can become expanded. So instead of having 36 repeats or a normal range between 18 and 50 repeats, you have 300 repeats. One example is Fragile X syndrome that you may have heard of, Huntington's disease. So you can have a variation that results in repeat instability and expansion or contraction of repeats. And that actually changes the gene expression or the gene sequence and causes disease. All right, so this slide is supposed to tell me that we're going to talk about variation. So there's all these chairs, and they're all orange. So they're all the same, right? No, not quite. They're a little different. They're all chairs, and they're all orange, but they have variation. So variation can be broken down into two grand categories. So this is a variation that Darwin observed, so a variation in phenotype. So what things look like on the outside and what are the characteristics that are observable. And then there's genotype variation, or what are the differences at the actual gene level. And we're going to go over that in a little more detail. The variation has its origins in the genotype most of the time. And there are consequences to variation. Sometimes there's no consequence. You can change a base. You can change the encoding amino acid. And you have no consequence whatsoever, because it really doesn't make any difference in the environment of that organism. You can make things worse for them. You can make it better. Worse is not good. Better is good in terms of evolution. And those differences may be environment dependent, which I may not have spelled right. We talked about evolution and then the phenotypic variation and disease as one category of phenotypic variation. Variation means it is found to vary. That's all it means. It's defined in the genome by reference to a standard, which may or may not be normal. So back in the Human Genome Project, the standard, the reference sequence that was generated was actually a composite of several individuals. But it was their sequence. And as I said earlier, it probably contained disease gene variants that were not considered normal. There are lots and lots of normal variation, much more normal variation. But there are variations that may not be normal in the reference sequence or the standard sequence. Variation occurs normally without regard to functional consequence, which we're talking about. So the variation happens due to replication errors. The errors, in general, they're not made because they need to be made. They're made because they happen. And then they're selected through evolution. And it's subject to selective pressures. And interestingly, a variation can occur, originate, in one member of a population. And then the descendants of that individual, at least some of them will inherit that and pass it on. So that results in frequency differences between different population lineages. Can anybody explain that better? Or give an example so somebody can try them. Go for it. It's kind of a founder effect, is that what you're kind of describing? So if you carry a disease and you're on an island in the Caribbean and you pass it to all your children or whatever, you have 10 kids, then that would then spread the disease. If it's like a single, is that what you're kind of saying? Yeah, another example might be populations that tend to stay together. Certain tribes or populations, the one that we think we hear about more often in medicine is Ashkenazi Jewish populations, who tend to. The Amish are a good group as well. So if that particular variant, which originated in that population, if that population is not strongly outbred, then it will remain associated with that population, have a higher frequency in that population than it does in another population that didn't have the opportunity to interbreed. OK. Any other questions? Yeah. To pass on any radiation for the next generation, does it have to be the reproductive cell or any other somatic cell can pass on the radiation too? Only the reproductive cells. So if it's not put into the sperm or the egg, it doesn't get passed on. So what you may be getting to is cancer mutations. So if a cancer mutation, we differentiate, I see this is a really good question, so we differentiate cancer mutations between those that are somatic, meaning they're in the body cells that can cause cancer, or those that are germline, meaning they're in all our cells, including the ones that contribute to egg and sperm. That get passed on. And it's the latter that are the form for hereditary cancer predisposition syndromes. So like BRCA1, BRCA2 is an example where you have a gene which predisposes you to breast cancer in all your cells. You get breast cancer, ovarian cancer, lower frequency other cancers because those are more sensitive to that particular mutation. But in your germ cells, they're going to get passed on at 50-50 chance to your offspring. Does that answer your question? Really good one. Yeah. I'm not sure what the process is to say not to worry about these or to worry about these and to test other people in the family. It's a really good question. And we actually struggle with this a lot. And we're doing a lot of work to try to sort out exactly what that process should be. And one of the things that we do is to kind of divide those variants that we get back on a genome report, on a panel report, into categories that say, this is how much we know about this particular one. So a lot of the BRCA1 and 2 mutations we've seen before, it's very clear that they're associated with an 80% risk of breast cancer, for example. And we can say, very clearly, that's a pathogenic mutation. That's a mutation or a variant that causes disease. There are others that seem to, if you look at the biology of it, they say, well, they stopped the protein synthesis, which we'll talk about later. But we really don't know for sure that they're associated with this condition. And those are called variants of uncertain significance. And we don't know what to do with those. We're hoping that as we gather more information, we will know which ones of those are normal variants because they're significantly, they're seen in a population that doesn't have a high risk for breast cancer, for example, OK. What information? When we do the testing, is that? How to say whether this is going to be a problem or not. So there's a wide range of information. So we're looking at whether the particular variant is predicted to cause a difference in the protein that is bad for the protein. Or it may be bad enough that the protein isn't even made, or it's only half of it is made, in which case it can't form into a full functional protein. We're looking for association with families who have passed this down through generations. And in every generation, there's been a high risk. Those who have that variant have a high risk for cancer. And those who don't have that variant don't. So it's a different kind of information, all right. We're looking in model organisms, which I think you're going to hear more about too, whether if we introduce that mutation into the model organism, is there an increased chance of cancer or whatever the phenotype is in that model organism? We're looking in chemical assays in the lab to say, if you introduce that mutation into cells in the laboratory, do they behave like cancer cells, for example, or do they do things that only cancer cells do, or make chemicals that only cancer cells do? So there's a whole wide range of information that's trying to be put together to make that determination. And then on the other hand, part of the precision medicine initiative is to sequence lots and lots of people, the whole genomes of lots and lots of people, and of all ages, and figure out which variants are out there that aren't disease causing. So we don't yet know completely what's the limit of normal, what variations are normal. So if we can definitively say that variation is not associated with cancer or anything else, then we can check it off. And we don't even have to report it to you or to the patient. Can I answer your question? Yeah. OK, so here's some other confusing points. And we're getting close to the hour, so I can't remember exactly where I am. So these are things that are not synonyms with variation. Mutation we used to use, and I still do because I'm an old fart, mutation we used to use to describe something that was different, especially in disease things. So a mutation is, but now we use the term variation for this very reason. Because a lot of times we're not sure whether it's a disease causing variant or not. But we call it a variation. So we reserve the term mutation now for the molecular and chemical processes that result in new variation. Otherwise, we try to avoid that. The other reason is because the term mutant, at least in high school, is not very well-received. So it has a negative connotation, and we try to stay away from that. Unless you like the X-Men. Unless you like the X-Men, that's right. So polymorphism is a term. Anybody run into that term before? Polymorphism is a single nucleotide polymorphism as an example. And it's a variation that exists in the normal population at 1% frequency or higher. That's all it means. And it's an old term based on population genetics. And it's called a polymorphism. It's assumed but not proven to have no disease significance, at least for rare diseases. When we get into more common diseases, all bets are off. And an example is the single nucleotide polymorphism. So one base is changed to another base, also known as a SNP. A new variation from mutation is altered between the last generation and this one. So somewhere in the production of the sperm or the egg, a replication error or something happened. And it's a new change that isn't in either parent, but it is in offspring. And that's called de novo, which is one of those dead languages for new. Marker. Remember, I showed you the map during the Human Genome Project? So markers are variations that were used to trace inheritance of DNA segments or suggest linkage disease genes. And the genome-wide association studies that you may hear about are an example of that and as well as linkage analysis, which is the old way we used to trace variations through individuals and through families. OK, so mutation, how does the mutation happen? What are the different kinds of mutation or variation that can happen? This is a mutagenic event. You have your double-stranded DNA with complementary sequence on both strands here. And the event affects this G. And a number of things can happen. It can be deleted. Then the other strand has to compensate. The C goes away. You can have an insertion. You can add a base, more than one base, as many bases as you want as an insertion. Or you can substitute a base from G to an A, for example. And then what happens when you replicate that? The C becomes a T. So that's sort of on the micro scale and on the macro scale. You can have big changes. These are chromosome diagrams, chromosome sticks. And you have a segment here that just gets deleted. And all the genes that are there get deleted. And the result is that instead of having two copies of this chromosome, you have one normal copy and one copy where some of the genes are deleted. And you only have one copy of those genes. That's important if the dosage of that gene matters for the instructions for running the human body. You can duplicate it, which is the converse of that. You can take that segment and flip it over, put it back in the same spot. And that's called an inversion. That usually doesn't cause a problem unless something at the break points at either end is messed up. So that break point was in the middle of a gene or puts a regulatory element that drives a gene next to something that it doesn't normally drive. You can substitute. So you put something from one chromosome onto another chromosome. You insert it. Important thing here is that everything here, inversion in the substitution, everything is in balance. The amount of genetic material and the copy number of genes is all the same. So that can happen without a lot of likelihood of there being a problem. Translocation is the same thing. You take something here and put it on one. And then the corresponding reciprocal change, this piece goes down here and you get that reciprocal. So in the individual holding that if it's absolutely clean without deletion or duplication at the break points, that may be OK. They may not have any disease. But when they pass that on, this variant is going to match up with its partner here. And the germ cells and the offspring have a chance of inheriting an unbalanced set of chromosomes. When should we take a break? OK, two more in this section, so we'll finish up this section. So genotype codes for phenotype. So I've been throwing these terms around, and so I'm trying to define them now a little bit late. So genotype is the genetic code describing an individual. It's that set of variations that we've been talking about that is unique to the individual. And the phenotype is the physical manifestations of genotype in an individual. This is sometimes a really difficult concept. For me, it's like falling off a log. I do this in my sleep, but I'm a geneticist. So maybe you can tell me if there are questions about these differences, genotype and phenotype. No, OK. In this example down here, we have the phenotype of fruit flies, normal wings, normal wings, normal wings, and wrinkled wings. How do you get wrinkled wings? In this case, it's because of a homozygosity for a recessive variant that's present on both copies, both the parental copies of the chromosomes. So the genotype in these two in the middle, who are heterozygous, is big W, little W. So there's one of the mutant alleles and one normal allele either way. That's their genotype, their heterozygous for the small W. But their phenotype is exactly the same as the homozygous for the large W. So it's a difficult concept. Think about it some. Maybe you've got it down. That's great. I wanted to make another point about variation and environment, which I think we've talked about. But here's I just pulled this Friday off of PubMed. And this is a krill. It's a tiny little organism that lives in the sea and is basically the bottom of the food chain for sea organisms. And what happens with climate change is that the seawater temperature changes. But this krill has been around for eons and is used to certain temperatures and is optimized for certain temperatures. So when you change the temperature of their environment, that creates a situation where it may not be optimal for that organism. So it may screw up the entire food chain. I'm throwing out some examples that your students might want to think about in terms of how this all impacts just everything around us. All right, more stuff about variation. It's essential. It's risky. It's relevant. And it's relentless. It happens all the time. And it's kind of like puberty, right? So OK. So I'm going to stop there to take a break. How many minutes? Five minutes?