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Video transcript

have you ever wondered how we sequence DNA let's just take a quick look at DNA sequencing so we're going to break down DNA sequencing into three different steps so the first step is you take the sample of DNA that you're interested in sequencing and you basically use PCR to amplify the sample so by using PCR in order to amplify the sample you're able to generate lots and lots of DNA fragments so the next thing that you do is normally in PCR you have to add nucleotides you have to give the growing strand the substrate from which it can grow so normally you add in regular deoxynucleotides and those look something like this got an H group here you've got an H group here you have a base and then you've got Carbon group and oxygen hydrogen so this is what a normal nucleotide looks like but interspersed in the PCR which you also want to add is you want to add in something known as a dye deoxy nucleotide so ad ID oxygen nucleotide looks something like this it's basically exactly the same thing but it only has a hydrogen here so this oxygen is removed and what that basically does is if this dye deoxynucleotide we can abbreviate DD ntp if this incorporates into the growing strand since there's no oxygen group here the strand can no longer elongate so you basically have termination of strand elongation as soon as this DD ntp incorporates so what you can do is you can actually fluorescently label the different dioxin nucleotides so for example we've got so for example we have four different options so we can label all the g's blue we can label all the a's red all the t's green and all the C's Orange and so basically what you have is you have these dioxin nucleotide different fluorescent labels getting incorporated into the growing strand and since PCR is able to amplify create millions and millions of DNA fragments you can basically what you can do is you'll have strands of different lengths so let's just kind of look at an example so let's imagine that we've got nucleotide be incorporated here a regular nucleotide and then another one incorporated here and then another one and then just randomly all of a sudden we have a dioxin nucleotide being incorporated here and this would stop elongation of the Strand so you'd have a DNA strand which is that's just four nucleotides long and after another round of PCR what we might have is we might have 1 2 3 4 5 6 it just growing it's growing it's growing and then all of a sudden whoa well happened you've got a dye deoxynucleotide be incorporated and so basically you just do this and after you've got millions of samples you'll eventually be able to have something that looks like this you'll have maybe just one regular nucleotide and you've got a dye deoxynucleotide incorporated or you might have maybe let's say to them so you'll have two and then you've got a two color so you've got a dye so what you can basically do is you can see that you have strands and they're in long eating and different strands are terminated at different points by a dye the ox nucleotide and so basically the next step is you use gel electrophoresis electrophoresis in order to separate the strains by size so when you run all the different fragments on a gel it'll separate them by size and then you can just have a computer go in and analyze all the fluorescent labels so if it sees here that you've got this blue fluorescent light then it knows that the second nucleotide in the in the sequence is a G so it'll say G and then it'll look here and it will say okay well this this is a C it will look here so we have another G and so on and so forth and basically the computer is able to by reading these fluorescent labels these fluorescent tags it's able to give you a DNA sequence and so this is basically the an overview of how DNA sequencing works
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