Polymerase chain reaction (PCR)
Introduction to PCR (polymerase chain reaction).
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- What exactly is a primer?(40 votes)
- A primer is a strand of short nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA (it needs a 3' OH to attach to). The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand.
In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA (which can be made de novo).(86 votes)
- In PCR, we use heat to separate two strands of DNA but we don't have enzyme or protein to keep them apart. How can we keep DNA single strand when we decrease the temperature for Annealing? I just quite confuse about this.(14 votes)
- He mentions it briefly in the video, in the liquid there are lots of DNA primers around, so when it cools down it is more likely the primer will attach to the DNA strand (and thereby blocking another DNA strand from attaching, rather than another DNA strand merging. I'm sure it sometimes happens that the DNA renatures before a primer could attach as well, but most of the time the primer will attach :)(28 votes)
- What's "taq polymerase"?(7 votes)
- The most important enzyme in a PCR reaction is called taq polymerase. A polymerase is an enzyme that attaches molecules together, and we just so happen to want to attach many nucleotides (the building blocks of DNA) together, so it works out for us. Every cell that has DNA has its own polymerase that takes care of replication of DNA. PCR uses a polymerase from a species of bacteria, Thermus(or thermophilus) aquaticus, which normally lives in hot springs.(17 votes)
- Why do you have to cool the separated DNA strands to attach the primers? Will the primers not bind to the DNA in a heated solution?(7 votes)
- Primers bind to longer nucleic acids by making base pairs. You can think of each base pair as being like the interaction between one hook and one loop in velcro. As you line up more correct pairs the interaction between the primer and its target sequence gets stronger, so longer primers tend to bind more strongly than shorter primers§.
The strength of a primer-target interaction affects how resistant it is to denaturation (i.e. separation of the two strands). This means that interactions between primers and a sequence they don't perfectly match will denature at a lower temperature.
Consequently, primers are designed so that they can only efficiently bind to a perfectly matched sequence at relatively high temperature — e.g. 55°C. This reduces the chance that the primer will bind to the wrong sequence.
Primers can be made that bind at the extension temperature (72°C), but longer primers are more difficult to make and thus more expensive. Primers around 20-25 nt long generally show good specificity and are relatively inexpensive, so that is what typically gets used. Primers in that length range typically bind best in the 50-65°C range.
§Note that the strength of interaction between G:C pairs is stronger that for A:T pairs, so the "GC content" also matters.(8 votes)
- Hi, I have a question. Here to make a single strand from double strand we are heating up the DNA. So, when transcription or any process is happening which leads to making a single strand inside our cell, is there any slight temperature rising in our cell or it is happening in just normal temperature?(6 votes)
- Interesting idea, but the separation happens without any significant heating. A cell would have to raise the temperature well over 90°C to have a significant effect on chromosomal DNA, so that probably would kill the cell assuming it had enough energy.
However, you aren't completely wrong — promoters contain A:T-rich sequences that are easier to separate ("melt") due to A:T base pairs having a weaker interaction compared with G:C base pairs.
The process seems to involve the RNA polymerase complex unwinding and forcing the strands apart. In eukaryotes this complex also temporarily "unpacks" the DNA from chromatin, so this is quite a complex process.
These activities open up what is known as a "transcription bubble" — a short (around 13 nt) region where the two strands of the DNA double helix are separated from each other. This bubble is moved along the DNA by the RNA polymerase complex as it transcribes the DNA.
(Replication follows a similar pattern, though of course using different enzymes.)(7 votes)
- I didn't understand why we need PCR, why we can't use the fragment of DNA we have, why we need more copies of the same fragment?
one fragment is not enough?(4 votes)
- To have enough DNA to clone or see on a gel you typically need billions of individual strands!
For example, in general you will need around 10 ng (a nanogram is 10⁻⁹ grams) of DNA to reliably see a band.
To convert grams to numbers of molecules you can use the average mass of a single nucleotide (remember to double this for double stranded DNA):
So, for a 100 bp fragment of double stranded DNA I get ~9.7 x 10¹⁰ molecules. Similarly, for a 1 kb fragment you would need ~9.7 x 10⁹ molecules.
Thus, if you don't use PCR to amplify the fragment, then you would need to extract DNA from billions of cells. This is possible, but then you would also need to separate the fragment you cared about from the millions or billions of other fragments that came from cutting up all the DNA in a cell.
With PCR you can instead make billions of copies of the piece of DNA you care about while the DNA you don't care about remains unamplified.
Does that help?(7 votes)
- We can seperate DNA strands with heat but why we can't basically use Helicase enzyme to seperate them?(5 votes)
- Because DNA would re-attach and form double-strand structure again.(3 votes)
- 1. How do people know what sequence to pick for their primer when ordering from a company?
2. Do the primers fall off each cycle or is there a polymerase that removes them and fills in those bases?(5 votes)
- 1. They have to do an experiment to find the exact primer to use.
2. There is a polymerase that removes them because a primer does not just fall off. If a primer falls of the reaction will not occur.(3 votes)
- So the point of PCR is to get many copies of a certain fragment of DNA. But why would you want to get only that fragment in the first place? And isn't it essentially the same as DNA cloning, where you cut certain gene from a DNA and make many copies of it? Or am I getting it wrong?(4 votes)
- PCR is one of many methods to increase the amount of a segment of DNA.
The reason we need to make multiple copies is that most of our current techniques rely on having 10's of billions (if not trillions) of molecules to work with!
Does that help?(4 votes)
- What is a plasmid?(3 votes)
- The plasmid is genetic material (part of DNA9 which is extra-chromosomal.
Small, circular, double-stranded DNA.
Bacteria are known for having plasmids but also some Eukaryotes have them.
- [Voiceover] 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? - [Voiceover] PCR was kind of the mainstay of my graduate project, where I built all sorts of different recombinant DNA molecules, and used them to learn things about plants. - [Voiceover] And so what does PCR in particular do? - [Voiceover] PCR basically makes you a lot of copies of a particular fragment of DNA that you're interested in. - [Voiceover] And so how does that... Why would you need to make a lot of copies of a particular fragment of DNA? - [Voiceover] 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. - [Voiceover] 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 sticking one fragment into one plasmid, you're doing that with many, so you need a lot of fragments of DNA. - [Voiceover] Exactly. That is exactly it. - [Voiceover] And you might start with a very small sample of DNA. Where else would you have to do PCR? - [Voiceover] 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 see if you have a gene that would predispose you to a particular condition, all sorts of really practical applications. - [Voiceover] Because it's hard to identify just one fragment of that gene. So you want to make copies, or as they say amplify it, so that you could run it in gels and stuff and see how all of those molecules, how big they are or something like that. - [Voiceover] 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. - [Voiceover] 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 double-stranded DNA, and this 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. 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 multiple copies of. And so this first step, denaturation... I have trouble pronouncing things. - [Voiceover] It's a weird word. - [Voiceover] 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 separates the two strands. - [Voiceover] Precisely. - [Voiceover] 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 one thing to remind ourselves is, this process is happening inside of a test tube, or in a big solution, So you heat it up, the DNA, the two strands separate, and do you just have this primer lying around? - [Voiceover] So the primer is something 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. - [Voiceover] So if this is our solution, is this all happening in water? - [Voiceover] Water with some salts and stuff floating around, yeah. - [Voiceover] 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, so let me do that in this magenta color. You obviously wouldn't see it in real life, it would just all dissolve. - [Voiceover] It would just look like a drop of liquid. - [Voiceover] It would look like... But for visualization, you'd put a lot of primer, and so you heat it 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. - [Voiceover] Exactly. - [Voiceover] And so when you order online or wherever 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. - [Voiceover] Exactly. That's super important. - [Voiceover] 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 it's called Polymerase Chain Reaction, that this is where the polymerase is involved. - [Voiceover] That is exactly where the polymerase comes in. - [Voiceover] So the polymerase is what is actually extending this. So I'll just draw a polymerase enzyme right over there, doing the extending, and is it any type of polymerase enzyme? Can I just take the polymerase from my cells and throw it in there? - [Voiceover] 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 polymerase is actually from a really heat-tolerant micro-organism. - [Voiceover] And what is that? It's called a taq polymerase? - [Voiceover] It's thermophilus aquaticus, I think? Makes quite a mouthful. - [Voiceover] And they found it at heated vents, this organism that is able to stand these high temperatures. But that I guess leads to another question, which is why do you have to heat it up to begin with? I guess just to separate the two strands? - [Voiceover] 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. - [Voiceover] Okay, so I get it. So this is one step, I'm getting at least the polymerase part of the PCR, where you heat it up, the strands separate, then you have all of this extra primer there. Because there's so much primer, the primer is much more likely to bind to, 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, the taq polymerase in particular, and you would have added that at the beginning, the taq polymerase, I guess I'll put it in a yellow color. So you would also put all that taq polymerase in there. And once again, these things aren't robots, they don't know exactly what they need to do, they just bump into things in the right way and react in the right way, and then you would also have to add a bunch of nucleotides. - [Voiceover] Yes, absolutely. Your reaction is not going to work if you forget the nucleotides. - [Voiceover] So, the taq 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 time? Will it just keep going on forever? - [Voiceover] 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. But it kind of will stop. Either it will fall off or it'll stop when you go on to the next step. - [Voiceover] Okay, so this, I get this so far. So, so far we have after one cycle, what you've written here, after one cycle we would have doubled at least that part of the sequence that we care about. Although we might have copied even beyond that sequence. So, where does the chain reaction come into this? - [Voiceover] So I guess you can interpret chain reaction in two ways, and one is that's sort of what the polymerase does, is you know, add 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. - [Voiceover] So you do it one cycle, you get to this situation right here, you heat it up, the strands separate, you cool it down, the primers attach, you heat it up again, the taq polymerase does its job, and like all polymerase, it goes from the 5 prime to the 3 prime direction, we talked about that in the application. - [Voiceover] Precisely. - [Voiceover] So now you have two strands, but now, since all of that stuff is in that solution, you can heat it up again, now these two strands can turn into... Or 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 they're still the same primer, because we still care about the same sequence. 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 typical for you to repeat this cycle? - [Voiceover] So like, 35 might be a pretty typical number of cycles to do. It depends a little what you're doing. But you're going to do it a lot of times. - [Voiceover] And so if you do this 35 times, I mean each time you're multiplying by 2. So it would be 2 to the 35th power, which is well over a billion times, so how long would that take? You've done this before. - [Voiceover] It depends on the length of your fragment, but usually like two to three hours. - [Voiceover] So in two to three hours, you can start with one fragment, and get into the billions. - [Voiceover] If it's perfectly efficient, which I wish it always were, but you usually get quite a few pieces made. - [Voiceover] And one thing that I've always wondered when I first learned about this, and I'd like to go into a lab and do this with you, is okay, I get that you have your primer, and then the polymerase is just going to extend it, like that, but I was like well, 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, when it goes in the other direction, it's going to hit a... It's not going to have anything else to copy. - [Voiceover] Exactly. - [Voiceover] So most of the billions of molecules that you produce, are going to be both ends kind of a nice clean cut. - [Voiceover] The vast, vast majority, exactly. - [Voiceover] Fascinating.