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Review 1: Nucleic Acids, Lipids, & Membranes

DNA Sequencing 1


DNA Sequencing

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you might remember from biology that DNA plum race actually requires a few things in order to synthesize DNA, including an RNA primer, a template strand and dioxin nucleotide triphosphate. It's it's gonna use that template strand to know the correct order basis to put together, because the template strand is going to have a base sequence that's complementary to the sequence that DNA Polyamorous wants to synthesize. And it's going to use the dioxin nucleotide. Try phosphates as the monomers for the polymer. And remember, it's going to be using the energy from braking those phosphate bonds in order to actually drive this reaction. Now it actually has to use an R in a primer. Because DNA plum race can't start synthesizing a new strand of DNA on its own, it actually can Onley elongate a strand that already exists. So it's going thio, uh, are in ourselves. Rather, we lay down a small stretch of RNA on the template strand, and then DNA plane race comes and attach is and lengthens that, of course it. It creates a strand of DNA, and later the RNA is removed. But you don't really need to worry about all that you just need to understand the basics of how DNA Plame race works. And you can see the reaction carried out by DNA plum race summarized in this equation right here where we have a primer of length and and a d oxy nucleotide triphosphate. And then the reaction occurs where the nucleotide is added and we're left with a primer of length and plus one, right, or it's basically have our primer and we've added one nucleotide onto it and a Z byproducts. From that reaction, we get pyro phosphate and a hydrogen ion. And those byproducts are actually going to become important later when we talk about some other DNA sequencing methods. But for now, we're going to talk about the O. G s DNA sequencing method, and that is Di di Oxy DNA sequencing and basically di di. Actually, DNA sequencing is a a method that relies on the's dia de oxy nucleotide triphosphate. It's like you see right here so normally a d oxy nucleotide triphosphate eyes missing a O. H. Group on the two prime carbon right there in Di di Oxy nuclear titrate phosphates, right? That's di di oxy. Like two d oxy. They're actually missing not only the two prime hydroxyl, but also a three prime hydroxyl group. Now, this is very important because during the course of DNA synthesis, we have, uh that three prime hydroxyl right? That is the attachment point for the next nucleotide. So in the course of the reaction, we actually add our new nucleotide on to that three prime hydroxyl, right? So we create a bond there. And then, of course, as we saw above are byproducts are hydrogen ion and this pyre phosphate right there. So if we don't have on O h group, right, if r o. H is missing on that three prime hydroxyl, we don't have anything to add a new nucleotide onto. So when a di di oxy nucleotide triphosphate is added to a new clinic acid, it's going to halt synthesis, right? It's going to cause the strand of DNA to stop at that particular nucleotide. So how is this actually used for sequencing? Well, in a very clever way, I would actually say, um, you know, it is a seemingly low tech method of sequencing DNA, but it's very sophisticated thinking behind this. So basically what you dio is you are going Thio run for DNA synthesis reactions. Right. So you're gonna and in each in each one you are going to use some sort of, you know, like Mila Melor, concentration of dioxin, nucleotide triphosphate, it's and then a very small, like micro Moeller concentration of die de oxy nucleotide triphosphate. It's so you're gonna have mostly the normal ones, right? Mostly the material that DNA polyamorous wants to use for synthesis. And then just a small amount of these di di oxy nucleotides that are going thio, of course, halt synthesis whenever there added. So basically, there's just a small chance. There's just a very small chance that you're going to add a diet e oxy nucleotide triphosphate, meaning that, you know, in the big vat you're carrying out this DNA synthesis in on Lee. Ah, small portion of the strands of DNA you synthesize are going to be truncated. Are are going to be shorter because of having added this diet e oxy nucleotide triphosphate. Now, as I said, you're actually going to do this reaction four times because in each one you Onley wanna add one type of the di di oxy nucleotide triphosphate. So you know let's say in reaction one, you do it with fining reaction to you Do it with cytosine. Three add ning and four wanting and you have some way of identifying what type of, um or you have some way of identifying you know which one is which. So you do them in separate are or you you You separate thes reactions so that you you only have one type of diet the oxy nucleotide triphosphate you're using, you know, just thio make it easier. Thio parse out which is which. So anyways, basically what you're gonna wind up with is ah, bunch of strands of different lengths from each DNA synthesis reaction. And you're gonna separate them using gel electrophoresis or something to determine the order of lengths and from the order of lengths you're going Thio be able to actually determine the sequence. So basically, you're going to see that. Okay, so you know your primer length right here we have our primer. You know how long the primer is, and then you see that there's a strand that came from you know, the reaction with Di di Oxy G, right, Like number four here. And that strand is one nucleotide longer than the primer. So you know that that's going to be a G. And then from that same batch of DNA, you have one. That's too, uh, you have a strand that's two nucleotides longer than the primer. So you know, that's also G. And then from Reaction one. You have a strand that's three nucleotides longer than the primer. So you know that's gonna be a T there and so on and so on and so on. So in this way you're actually able to determine the sequence of the DNA. And it's a very roundabout way of determining the sequence. If you think about it right, which is why I say it's a very there's, You know, it's not the most high tech method, but there's very sophisticated thinking behind this, all right, so let's actually turn the page and talk about a specific or some specific ways in which this is used