Positional Cloning

Hi in this video we're gonna talk about positional cloning. So positional cloning is an experimental approach that takes a list of candidate genes or a specific region on a chromosome and attempts to identify which one is the one of interest. So most commonly they're asking the question which one is causing a disease. So you have some kind of disease phenotype. You know, there's a section of a chromosome that's causing that's responsible for that. But what region and what specific gene is responsible for that phenotype. So here are the first five steps to positional clothing. The first step is you use traditional mapping techniques either through pedigrees if you're working with humans or through um actual crosses. If you're working with organisms you can make in a laboratory setting. But essentially the first step is identifying the chromosome region that's responsible or associated with the disease. And you do that through mapping. And so mapping is responsible for linking DNA markers and disease genotype. So we know we have a disease and we study the competence and the recombination frequency and that allows us to determine which chromosomal region between which DNA markers um is the gene that's responsible for that. Now these this region can be huge, It can cover hundreds of genes in it. But it is the first step of identifying, you know, what region of the entire genome are we looking at? The second step is once you have this region, you finally have a region could have 100 genes that could have five. It doesn't matter how long it is. But essentially you've narrowed it down. You're focused in on a region. The second step uses additional DNA markers within the low the low side to precisely Locate. So if you have this sequence right here and you know it's in this region now this could have 100 genes. Could have 10. It doesn't matter. You take DNA markers and you say there's one here, there might be another one here here here and here and you start dividing up this region with DNA markers. And so then you can look between each one of these markers and say well you know which one is causing the disease? Is it between this marker and this marker or is it between this marker and this one. And so that's the second step is sort of narrowing down using additional markers. Using additional mapping. Looking at the recombination frequencies to narrow down which specific region. So once you've identified two markers. So we'll say this one and this one and you know the diseases in in in this region use a technique called chromosome walking and what this is is it takes overlapping D. N. A fragments um and it identifies the gene of interest. So what happens is generally you have some kind of genomic library which if you don't remember what a genomic library is the video on it. But just as a refresher, a genomic library is just a bunch of short overlapping fragments of DNA from the genome. So you have a genomic library and you find one you find a particular clones or a particular sequence that fits here. So we'll say this is cloning and this one is the closest you can find that exists between this marker. Then you look through your all of your sequence and you'd identify another clone, clone B. And this clone overlaps with clone A. And you keep doing this right. So then you have clone C. And you do it as many times as you need. All the way through Z. And all the way back again until you identify where the gene is. So you repeat this chromosome walking as many times as you need sort of walking step by step towards the gene, one clone at a time. And so in this region that you finally identifies a clone. See here and here in remember you're just getting smaller and smaller. So now we're between clone C. There could be one gene, it could be a small number of candidate genes. But essentially we started out with the gene could be in the entire genome. But now we've narrowed it down to a short region of candidate genes or one particular gene. So you say, okay, well let's say there's three genes in here this uh clones, see that we've identified. So what you do is then because three is much easier to work with. And potentially 20,000 you take each gene individually and look at different phenotype that it causes. Where in the cell or in the body. Is it expressed when is it expressed? Could it potentially at all be associated with this disease? And through these additional examinations, which is step five, we can actually determine which one is that 12 or three causes the Vienna type. So let's just say we looked at gene to it was expressed where the disease is caused. Um and it's expressed at the right time. So gene two is the gene of interest. So positional cloning, you start with potentially the entire genome. You do some mapping. You use DNA markers and chromosomal walking to sort of narrow down on the whole genome to identify the specific gene or genes that's responsible for causing the disease. So here's kind of the first few steps. So the first thing you start out is napping. So in humans we can't do crosses, right? So we just have to deal with the crosses that are already done. So in this case this is we use pedigrees for human crosses. So here's a pedigree and we take DNA segments from every single person in this family. And probably even more than what's shown here. And through looking at recombination frequencies of different DNA markers, we determine that this region here is responsible for this disease, whatever it is, doesn't matter. So from there we take it and we take it a little further we either do more markers. We can do chromosome or walking. But eventually we find this gene and this gene is responsible for this disease. And we totally just did it through mapping of different regions through positional cloning. Taking a step by step going. Starting out very large with a pedigree, doing mapping, looking at recombination frequencies and markers to get here and then you keep repeating it and keep repeating it chromosome walking. If you need to eventually identify the gene of interest that's actually causing this disease. So positional cloning, the best way to think of it is going from pedigrees to the gene and that is how that is done. So with that let's not move on.