DNA Sequencing 1 - Video Tutorials & Practice Problems
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DNA Sequencing
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You might remember from biology that DNA polymerase actually requires a few things in order to synthesize DNA, including an RNA primer, a template strand, and deoxynucleotide triphosphates. It's going to use that template strand to know the correct order of bases to put together because the template strand is going to have a base sequence that's complementary to the sequence that DNA polymerase wants to synthesize. And it's going to use the deoxynucleotide triphosphates as for the polymer and remember, it's going to be using the energy from breaking those phosphate bonds in order to actually drive this reaction. Now, it actually has to use an RNA primer because DNA polymerase can't start synthesizing a new strand of DNA on its own. It actually can only elongate a strand that already exists. So it's going to or in our cells rather, we lay down a small stretch of RNA on the template strand and then DNA polymerase comes and attaches and lengthens that. Of course, it it, creates a strand of DNA and later the RNA is removed but you don't really need to worry about all that. You just need to understand the basics of how DNA polymerase works. And you can see the reaction carried out by DNA polymerase summarized in this equation right here, where we have a primer of length n and a deoxynucleotide triphosphate. And then the reaction occurs where the nucleotide is added and we're left with a primer of length n+1. Right? Or it's basically we have our primer and we've added 1 nucleotide onto it. And, as byproducts from that reaction, we get pyrophosphate and a hydrogen ion. And those byproducts are actually going to become important later when we talk about some other DNA sequencing methods. But for now, we're going to talk about the OG's DNA sequencing method and that is dideoxy DNA sequencing. And basically, dideoxy DNA sequencing is a a method that relies on these dideoxynucleotide triphosphates like you see right here. So normally a deoxynucleotide triphosphate is missing a Oh group on the 2 prime carbon right there. In dideoxynucleotide triphosphates, that's dideoxy like 2 deoxy, they're actually missing not only the 2 prime hydroxyl but also a 3 prime hydroxyl group. Now, this is very important because during the course of DNA synthesis, we have that 3 prime hydroxyl. That is the attachment point for the next nucleotide. So in the course of the reaction, we actually add our new nucleotide onto that 3 prime hydroxyl, right? So we create a bond there and then of course as we saw above, our byproducts are hydrogen ion and this pyrophosphate right there. So if we don't have an Oh group, if our Oh is missing on that 3 prime hydroxyl, we don't have anything to add a new nucleotide onto. So, when a dideoxynucleotide triphosphate is added to a nucleic acid, it's going to halt synthesis. It's going to cause the strand of DNA to stop at that particular nucleotide. So, how is this actually used for sequencing? Well, in a very clever way, I would actually say. It it is a seemingly low tech method of sequencing DNA but it's very sophisticated thinking behind this. So basically, what you do is you are going to run 4 DNA synthesis reactions, right? So you're going to And in each in each one, you are going to use some sort of, you know, like millimolar concentration of deoxynucleotide triphosphates and then a very small like micromolar concentration of dideoxynucleotide triphosphates. So, you're going to have mostly the normal ones. Mostly the material that DNA polymerase wants to use for synthesis. And then just a small amount of these dideoxynucleotides that are going to of course halt synthesis whenever they're added. So basically there's just a small chance, there's just a very small chance that you're going to add a dideoxynucleotide triphosphate meaning that, you know, in in the big vat you're carrying out this DNA synthesis in, only a small portion of the strands of DNA you synthesize are going to be truncated or are going to be, shorter because of having added this dideoxynucleotide triphosphate. Now, as I said, you're actually going to do this reaction 4 times because in each one, you only want to add one type of the dideoxynucleotide triphosphate. So you know, let's say, in triphosphate. So you know, let's say, in reaction 1, you do it with, Thymine. Reaction 2, you do it with cytosine 3, adenine and 4, guanine. You have some way of identifying identifying identifying, you know, which one is which. So you you do them in in separate or you you separate these reactions so that you, you only have one type of dideoxynucleotide triphosphate you're using, you know, just to to make it easier to parse out which is which. So anyways, Basically, what you're going to wind up with is a bunch of strands of different lengths from each DNA synthesis reaction and you're going to separate them using like gel electrophoresis or something to determine the order of lengths and from the order of lengths, you're going to be able to actually determine the sequence. So basically, you're going to see that primer we have our primer. You know how long the primer is and then you see that there's a strand that came from, you know, the reaction with dideoxy G, right? Like number 4 here. And that strand is, one nucleotide longer than the primer. So you know that that's going to be a G. And then from that same, you know, batch of, DNA, you have one that's 2. You have a strand that's 2 nucleotides longer than the primer. So you know that's also G. And then from reaction one, you have a strand that's 3 nucleotides longer than the primer. So, you know, that's going to be a T there and so on and so on and so on. So in this way, you're actually able to determine the sequence of the DNA and it's a very roundabout way of determining the sequence if you think about it, right? Which is why I say it's a very there's you know it's not the most high-tech method but there's very sophisticated thinking behind this. All right. So let's actually turn the page and talk about a specific or some specific ways in which this is used.