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Polymerase chain reaction (PCR)

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Video transcript

I'm here with Emily our biology content fellow to talk about PCR or polymerase chain reaction which you've actually done a lot of why have you done PCR PCR was kind of the mainstay of my graduate project where I built all sorts of different recombinant DNA molecules and use them to learn things about plants and so what does PCR in particular do PCR basically makes you a lot of copies of a particular fragment of DNA that you're interested in and and why and so how does that like why would you need to make a lot of copies of a particular fragment of DNA so you might want to be making lots of copies so that you can clone it into a plasmid and then do some other experiments with it that's a big use so when we talked about cloning and we're talking about sticking a fragment of DNA inside of a plasmid it's not like you're just taking one fragment into one plasmid you're doing that with many so you needed a lot of fragments of DNA exactly that is exactly it and you might start with a very small sample of DNA and so you just really need to read where else would you have to do PCR PCR is used a lot in forensics it's also used a lot in medical diagnostics so this could actually be your DNA that was being checked to to see if you have a gene that would predispose you to a particular condition all sorts of really practical applications because it's hard to identify just one one fragment of that gene so you'd want to you want to make copies or as they say amplify it so that you can run it in gels and stuff and see how all of those molecules how big they are or something like that exactly if you were just looking in your DNA pulled out of your cell that would be a needle in a haystack so this is how you can really zoom in and look at just the thing you need to see okay so you've drawn some diagrams here and I actually have never done PCR but you have so I'm going to tell you how I understand it happening and then you tell me if this makes sense so what you drew over here this is this is double-stranded DNA just you know and could have been from a sample of someone's hair or whatever else and let's say we want to replicate or make many many copies of a fragment of this and so let's say the fragment that we really care about is the fragment roughly from there to there this part is what we want to make we want to make multiple copies of and so this first step denature ization denaturation denaturation have trouble pronouncing this weird word it's a weird word you have 96 degrees Celsius so this is almost at the boiling point so it's quite hot and that that separates the two strands precisely and so once they're separated then you can cool things down although this still isn't that cool 55 degrees Celsius would be very uncomfortable but you would cool it down to this and then these primers show up and so you know one thing to remind ourselves is this process is happening inside of a test tube or in a big solution so you you heat it up the DNA the two strands separate and you just have this primer lying around so the primer is something that that you've ordered from a company and you've ordered a lot of it so you put in a ton of primer in your reaction so that there's a really good chance that when you get to this step here called annealing that a primer is going to bind to many of your pieces of DNA so this is our this is our solution is this all happening in water water with some some salts and stuff floating around yeah okay so we have our solution right over here you'd put whatever your initial DNA sample is in there and once again it's a very small amount you'd put a lot of that primer so you'd want to put that in a lot of surplus let me do that in a blue this magenta color you obviously wouldn't see it in real life it would just all dissolve it would just look like a drop of liquid it would look like but for visualization but you put a lot of primer and so you've heated up the DNA strands separate and then when you cool it back down this primer is going to be specific to the ends of the region that you want to copy exactly and so when you go to the when you order online or ever that you want a certain primer you're going to pick the sequence of that primer to be specific to the regions you want to copy exactly that's super important okay and so when you cool it back down the primer attaches and then you heat it back up not quite to the 96 degrees Celsius but to the 72 degrees Celsius where you extend those and I'm assuming since we're it's called polymerase chain reaction that this is where the polymerase is involved that is exactly where the polymerase comes in so the polymerase the polymerase is what is actually extending this and is it so I can I'll just draw a polymerase enzyme right over there doing doing the extending and is it any type of polymerase enzyme could I just take like you know the polymerase for my cells and throw it in there so you actually need a special polymerase because you need one that is going to be pretty heat resistant so as you were mentioning even the cool step of this process is not something that your body would want to be hanging out in so the the polymerase is actually from a really heat tolerant microorganism and and what what is that it's called a tack polymerase thermophilus aquaticus I think makes quite a mouthful and they founded it at heated vents this organism that is able to stand these high temperatures but but that that I guess leads to another question is why well why do you have to why do you have to heat it up to begin with I guess just to separate the two strands that's really the key reason you just have to get them apart you don't have an enzyme to do it the way you might in a Cell so heat does the trick okay so I get it so this is one step I'm guessing I'm getting at least the polymerase part of the of the PCR where you heat it up the strands separate then you have all of this extra primer there the primer because there's so much primer the primer is much more likely to bind to at the at least at this part of the sequence then for these two strands to get back together at this point and then you have the polymerase to tack polymerase in particular and you would have added that at the beginning you know the tack polymerase I'll guess I'll put it in this I'll do it in a and a yellow color so you would already you would also put all that TAC polymerase in there and what say you know these things aren't these things aren't robots they don't know exactly what they do they just bump into things in the right way and react in the right way and you also have to add a bunch of nucleotides yes absolutely your reaction is not going to work if you forget the nucleotides so the attack polymerase when you heat it back up again after the primers have been attached is going to start adding all of these nucleotides and what do you just wait a certain amount of times it will just keep going on forever it'll keep going on for a while usually you do pick the length of that step to match how much time you expect the polymerase to need to complete your fragment on but it kind of will stop either it'll fall off or it'll stop when you go on to the next step okay so this I get this is so far so so far we have after one cycle would you've written here after one cycle we would have doubled at least that part that part of the sequence that we care about although we might even have we might have copied even beyond that sequence so what where's the chain reaction coming to this so I guess you can interpret chain reaction in two ways and one is that's sort of what the polymerase does is it you know adds things to make a chain but there's actually even more of a chain reaction to mention here and that's that we're actually getting this kind of exponential process going on so you do it one cycle you get to this situation right here you heat it up the Strand separate you cool it down the primers attached you heat it up again the tack polymerase does it's does its job and I call polymerase it goes from the 5-prime of the three prime direction we talked about in that very location so now you have two strands but now since all of that stuff is in that solution you can just keep you can heat it up again now each of these two strands can turn into or these two these two double strands can now turn into four single strands then you can cool it down again now they get primers attached to them and there's still the same primer because we still care about the same sequence and then that can key and so now you go from one to two to four and so you keep repeating this and so how many times would it be tip for you to repeat this cycle so like 35 might be a pretty typical number of cycles to do depends a little what you're doing but you're going to do it a lot of time and so if you do this 35 times I mean each time you're multiplying by 2 so it'll be two to the thirty fifth power which is well over a billion times so and how long would that take you've done this before um depends on the length of your fragment but usually like two to three hours so in two of the hours you can start with one fragment and get into the billions if it's perfectly efficient which I wish it always worked but you usually get quite a few pieces made and one thing that I was that I've always wondered when I when I first learned about this and I'd like to go to a lab and do this with you is it is okay I get that you have your primer than the and then the the polymerase is just going to extend it like that but I was like well it's you know it's going to be how does it know where to stop and you explained well on that first pass it might not know where to stop but then when you start going in the other direction it's going to so over here and when it goes in the other direction it's going to hit a it's going to hit a it's not going to have anything else to copy exactly and so then you're so most of the pull of the billions of molecules that you produce are going to be both ends kind of a nice clean cut the vast vast majority exactly fascinating
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