Hi. In this video, I'm going to 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 a DNA molecule. So that's, you know, what is the order of a, t, c's, and g's? You know, what order they are that makes a gene?
The first method that was created in the 1970s is called the dideoxy method. You may also see this as Sanger sequencing. They're the exact same thing. But, essentially, how is the basics of this is through using these special nucleotides called dideoxynucleotides, and they're abbreviated DDNTP. And the n stands for either a, t, c, or g. So essentially, these nucleotides are created without a special group, the 3 prime hydroxyl group. And this group, if you remember from DNA replication, is super important because it's required for nucleotide addition. So if a dideoxynucleotide gets added during DNA replication, then it 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 or 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 DDNTPs. And these, when they're incorporated, which will be rarely because they're at low concentrations, but when they 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 DNA. But the difference between this and normal PCR is that because you use these DDNTPs, you're going to have a bunch of copies of the same sequence, but they're all going to be stopped at different nucleotides. Because any time a DDNTP was added, the replication is going to stop.
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 DDNTPs also contain some kind of dye, fluorescence or otherwise. So then, you can just look at this gel, image it for those dyes, and the sizes will dictate how many nucleotides there are, and the color will dictate what nucleotide it is.
So, if we're looking at this, so here we have a DNA sequence and we're performing some kind of replication, PCR or otherwise. So what you can see is that we have our 4 DDNTPs. You have t, which is in pink. You have a, which is green. You have g, blue, and c, red. So when we do our PCR, some of the most of the nucleotides are going to be added just normally, but every once in a while, a DDNTP will be added, and that will stop. 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'll 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 colors for this? And you can see, if you look at the sequence, you can get TACGCGCAT, 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, sometimes this is still used, but it is much more rare. There are all these different techniques. I am not going to go into them as for a more advanced biology class, but it's always good to sort of look back at history and see, you know, how did we first start sequencing the genome and different genes. This is it. So with that, let's now move on.
