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Cell Biology

Learn the toughest concepts covered in Cell Biology with step-by-step video tutorials and practice problems by world-class tutors

22. Techniques in Cell Biology

DNA Sequencing


DNA Sequencing

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Hi in this video, I'm gonna be talking to you about DNA sequencing. So, DNA sequencing today includes a ton of techniques but essentially the purpose of all of these is to identify the nucleotide sequence of the DNA molecule. So that's you know, what is the order of 80 season G. S. You know what order they are that makes a gene. So the first method that was created in the 19 seventies is called the di di oxy method. You may also see this as sanger sequencing, they're the exact same thing. But essentially how is the basics of this? Is they're using these special nucleotides called di di oxy nucleotides and their abbreviated D D N D P. And the N. Stands for either A T C. Or G. So essentially these nucleotides are created without a special group that three prime hydroxyl group. And this group, if you remember from DNA replication is super important because it's required for nucleotide edition. So if a di di oxy nucleotide gets added during DNA replication then it's can't add any more nucleotides replication is going to stop right then. So what you do is you start by performing some kind of DNA amplification usually through a pcR PcR like method and you have the majority of normal nucleotides, right? The majority of everything in there is normal. But you also have a low amount of these D. D. N. D. P. S. And these when they're incorporated which will be rarely because they're at low concentrations but when they are are they will stop the replication. So like I said here, the addition of this would prevent further elongation of the sequence now because it's at low concentration, it's not going to be added often, but it will eventually be added. So then once you do that pcr a bunch of different times, you're going to have all these different copies of D. N. A. But the difference between this and normal PcR is because because you use these D D N. D. P. S, you're going to have a bunch of different copies of the same sequence but they're all going to be stopped at different nucleotides because any time a D D N D P was added, the replication is going to stop. So you're going to have the same molecule but it's going to be all different sizes. So then when you run this on some type of gel, you have many different sizes and you can see them. And the reason how you can use this for sequencing is because the D D. N. T. P. S also contain some kind of dye fluorescence or otherwise. So then you can just look at this gel image it for those dies and the sizes will dictate how many nucleotides there are and the color will dictate what nuclear it is. So if we're looking at this. So here we have a D N A sequence and we're performing some kind of replication PcR or otherwise. So what you can see is that we have our four D. D. And T. P. S. You have T. Which is in pink. You have a which is green you have G blue and see red. So when we do our pcR some of the most of them nucleotides are going to be added just normally. But every once in a while a D. D. N. D. P. Will be added and that will be stopped. So then when you run these on some kind of gel you'll be able to tell the size that it stopped and the nucleotide it stopped at because it will be represented by color. So if these were the fragments here we had pink green, pink, blue, red, red, blue red green. Then we can just look here and say okay well what are the what are the colors for this? And you can see if you look at the sequence you can get T a g c c g c a and that is going to be the sequence for this fragment. So that's super important. And that's how really the first way that we were able to do DNA sequencing now today more advanced technology is used. I mean this is still used but it's much more rare. There's all these different techniques. I'm not going to go into them. That's far more advanced biology class but it's always good to sort of look back at history and see how did we first start sequencing the genome and different genes. This is it. So with that let's now move on.

ddNTPs are unique because they do what?