 Although as interesting as single nucleotide polymorphism is we can really only go after a single size So we're already aware of and targeting and what we rather would like is to be able to study entire genomes sequence everything That is difficult though It's very difficult because again the molecules are too small and we have a sea of molecules and we don't really know where to start Do you remember Fred Sanger? I spoke about him very early in the class that he was the first one to determine the sequence of insulin and got the Nobel Prize for that. Well, Fred is one of the greatest scientists of the 20th century. He died a few years ago But he came up with another very smart methods in the 1970s that if you pretty much just run your PCR Remember the Kerry-Mollis method to amplify DNA, but instead of the normal basis A, G, C and T you mix them with some screwed up bases What kind of basis this so this is a A Base there are two deoxy so it's di deoxy. Do you remember that you had RNA and DNA? So ribonuclease and deoxy ribonuclease. So this is a di deoxy ribonuclease You don't have either of the oxidants here That in particular will mean that this base can't really make a link to the next base So if we incorporate this base, it stops there. We can't get further. That seems remarkably stupid But Fred came up with something really smart here This is a bit much to draw and I don't expect to know this by heart. So I'll I'll use a an illustration of it So that if we start from the sequence out there and then we have this C of Normal amino acids and let's mix in either. I only mix in one type of this one Or I mix in all four What's now going to happen is that when we start this process It will be random right that occasionally I will pick one of these bad bases by mistake And in that case I will just get a short fragment and then it will stop. I will not get further So assuming that I have 10 residues here if I have a dd ntp here that matches base one I will literally just get a segment of size one now and then if I hand on that matches Residue two I'm going to get segments of length two, etc And then it's in principle that means that I'm going to have these are roughly of size one two three, etc All the way up if I now put those on a gel a gel is a chemical way that I I add electrophoresis and try to pull the molecules slowly down through a yell And that means that the small molecules will move very fast So I will get them at the bottom while the heavy ones will stay at the top I'll skip those for a second and show the other way to do it Here we've done this for four separate cases In the first one here. I only had the a adenine base marked And then here at the bottom so at the bottom we start those are the smallest fragments that went all the way So I had something very small that might have been a and then I don't have anything and then I don't have anything And then I have an a again and maybe nothing and then an a again In the second column, I've done the same thing But now I've may now I marked the G's with a radioactive isotope And then I've marked the C's and then I marked the T's so here. I literally had to repeat the experiment four times But once you've done that We literally have the sequence here, right? So we can if we now read these bands in order and I'm well aware that it's going to be a bit difficult But if we read these carefully you can actually say what is the exact Abena not amino acid. What is the exact base sequence in my DNA strand here? In practice today, this is what we would do if we did it manually nobody does this manually today What you instead do is that you use fluorescence But with fluorescence we can control the wavelength. Remember things like the green fluorescent protein So if I now have agc and t Screwed up markers that will kill the polymerization. What if I mark them with four different colors? If I do that I just have to do one band here and then I can rather than doing radioactive marking on films and everything I can just have a laser Excite them and then detect the fluorescence here super quickly with the photodiode So then depending on what color I have the next base is either agc agc agc And then I'd better devise a machine that could do this pretty fast and trust me people have Fred got a second Nobel Prize for this so-called chain termination sequencing chain termination Sequencing the reason why I had nobody called at least I never called this chain termination sequencing We just call it Sanger sequencing after Fred