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Review 1: Nucleic Acids, Lipids, & Membranes

DNA Sequencing 2


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di di Oxy sequencing was pioneered by Frederick Sanger. And so, uh, his particular method is referred to as Sanger Sequencing, and this is basically died. The Oxy sequencing, uh, were used these radio labeled di di oxy nucleotide triphosphate radio labeled, meaning they have some sort of radioactive tag on them so that you can identify them. And, you know, just like we talked about you use, uh, you know, a much smaller concentration of the di di oxy nucleotides compared to the dioxin nucleotides. So that on Lee, you know, a small amount of the strands are truncated or shortened during synthesis. And, of course, you know, you only use one type of di di oxy nucleotide triphosphate per synthesis so that you know, uh, you know which which base, Um, which bases present when you find a shortened strand? Because if there's only one type of di di oxy nucleotide that you were using in the synthesis, anytime you find a shortened strand, you know that the last nucleotide on that strand is going to be whichever one you were using in that particular reaction. So then you actually use gel electrophoresis to separate the fragments of different lengths, and you can see that going on right here. This is our gel. And you can see that are fragments from our reaction with G are found here, here, here and here and that are fragments from Reaction Sea or along this path A along this path and t along this path. So, uh, if you were Thio actually try to read this code, you'd see that the actual code here is going to be something like a t g c T t uhh! Sea G. And I'm gonna stop right there. So this G is corresponding to this mark right here. So all I'm doing is reading down, reading down the gel, more or less to determine the code now. And, of course, uh, I know this to be the order because the smaller strands will travel further in the gel. And, of course, um, this arrow going up this way shows us the direction of travel. Eso the strands down on this end right here are going to be the smallest, and the biggest will be at the opposite ends. You just read from smallest to biggest, and that's your sequence. Um, eventually thes radio labeled dioxin nucleotide triphosphate Swor replaced with fluorescent ones. And the reason is, um, because A it's like easier, more or less in the lab. Thio. Try Thio scan for the light emitted from fluorescent molecules as opposed to trying thio work with radioactive substances. Also, you kind of usually have to use some pretty nasty chemicals when you're working with these radioactive substances. So, uh, it's just it's more efficient and you don't have to use thes nasty chemicals. And that's why there was this transition Thio using fluorescent ones but basically the exact same concept that's going on. Except instead of using gel electrophoresis, use a cap Hillary gel column So it's kind of like a electrophoresis gel, except you're running like you're running it in a column and your eluding out the various strands. And as the strands pop out right, smaller ones, they're gonna come out first. The bigger ones later. And as the strands pop out, you have a photo detector that's going thio. Uh, you know, you know, scan and determine what you know. Frequency or wavelength fluorescence. Each each strand is emitting, and based on the color that they're admitting it's going to determine which labeled di di oxy nucleotide triphosphate, uh, you have present. So you know, much in the same way we went through the gel. Basically, if we were the spectrum Tom Attar we'd read, like, as the strands come out would be like green read. Yeah, you know, black blue, whatever. And we know that green means a red means t black means g. I know it's yellow over here. So, you know, uh, the image isn't perfect. I'm sorry. And you know, this blue one would be see, But you get the idea. It's basically the same concept. Just the mechanics of it are slightly different because we're looking for for fluorescent light rather than looking for these radioactive molecules. All right, so remember previously when talking about DNA prelim race, we said that the by products of the reaction would become important for later sequencing methods and thes later sequencing methods include piras sequencing, which is sometimes called 454 because of the place that developed it. But, uh, you know, we're just gonna call a pirate sequencing because that's more like generic name for it, and basically pyre sequencing looks for the pyro phosphate released by DNA polyamorous in order to determine sequence so more or less you have thes beads in these wells in this big plate and this big plate with lots of wells in it and each well has a bead in it. And on these beads are sequences of DNA, and, uh, in addition to these beads with these sequences of DNA attached to them, you also have these enzymes and enzymes are self fearless and luciferase and basically, um, so fearless Will, uh, so fearless will react with pyre phosphate and generate a teepee. And so, if you have a nucleotide that gets added to your chain, you're going thio release pyre, phosphate and so, so fearless is going to make ATP. And because luciferase is there, if you have a teepee present, the luciferase is actually going to use that ATP toe light up. So, um, so basically, what is going toe happen is you have this, you know, big tray, all these wells in it, and you're going thio in each well, like one at a time. You're going to you add, uh, a particular nucleotide triphosphate. Right? And you're just gonna add them or you're going to add them to the well in sequence. Basically. So you're gonna add add means Look for light. You're gonna add diamonds. Look for light. Guan means look for light cytosine look for light and basically you're looking for light because if you see light, that means that the nucleotide triphosphate that you just added to your well was attached to your strand of DNA. It was, um it was attached to your growing strand, and it released the pirate phosphate. And that pirate phosphate, uh, was picked up by sulfur list added to a t p. And that a teepee activated luciferase, which is a a nen sign that gives off light. So basically, you repeat this process a ton of times. Um and you know, you're not doing this by hand. You do this in a machine, and it actually does it quite efficiently and basically looks for light peaks. And based on those light peaks, it is able to determine the sequence of DNA and, uh, in a similar concept to pirates. Sequencing is ion torrent sequencing And again, it's very similar idea to piracy sequencing, except instead of instead of looking for light flashes due to the release of pyro phosphate. Yeah. Looks for changes in ph due to the release of protons. Remember, pyro phosphate is released, uh, in, um, in conjunction with the proton. So I m torrent secret sequencing is basically looking for those proton releases instead of the PIRA phosphate releases, and it measures those proton releases by measuring ph changes. So very similar concept. But again, the mechanics are a little different. All right, let's turn the page.