Polymerase Chain Reaction - PCR - Video Tutorials & Practice Problems
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concept
PCR
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Hi in this video, I'm gonna be talking to you about preliminary chain reaction or pcR. So pcR is a method of amplifying D. N. A. So essentially the basic of it is is that includes multiple cycles, usually 25 to 40 of these three steps. So the first step that happens is you have A D. N. A. You want to amplify and you expose it to high temperatures. And this separates those two strands of the DNA double stranded. But in order to replicate it, you gotta separate them. And so the high temperatures breaks those hydrogen bonds and separates out the D. N. A. So this usually happens at 95 degrees Celsius. Then after you separate the D. N. A. That's super fantastic. But you need enzymes to be able to work. And typically they don't work at this high temperature. So then you lower the temperature. Um And um the first thing you do is you lower it to like 55 to 70 degrees somewhere in there. And what this does is allows for primers to bind water. Primers, primers of small nucleotide sequences that are complementary to the D. N. A. You're trying to replicate. And so they will bind to that D. N. A. At this lower temperature. And that says, hey D. N. A polymerase come here because this is the sequence you're going to replicate. So scientists design these primers because those primers say, okay this is the sequence, this is the start site, this is the stop site. This is where you're going to replicate and then you usually raise this to 78. It can be only around there and depending on what you're doing but essentially a D. N. A polymerase is added and then that can begin replicating the DNA. So then this is the third step. So once you do that for a little while you start back so you get multiple trials at 25 to 40 X. Cycles of these three steps. And um that allows to generate a ton of copies of D. N. A. So why do we do this? Um Well typically I mean it designs billions of copies of this D. N. A. Essentially. So why do we need that? Well scientists can use it in diagnosing diseases or in forensics applications like figuring out who D. N. A. Evidence of the crime. It's important for scientists who need to compare DNA molecules because if you have one DNA molecule you're not going to have enough information to be able to actually examine that. So you need usually multiple copies of it like a ton of it in order to be able to use technology to figure out what sequence it is and how it compares. You can use this technique to test for the present. So you say, okay well I want to know if this DNA sequences here, Let me see if I can amplify it if I can it will amplify there'll be tons of it if I can't it's not present. And then there are some new newer technologies that allow you to actually quantitative calculate the exact amount of DNA present in a sample. So D. N. A. It's called Q. PcR. This is quantitative PcR if you start with R. N. A. Typically do R. T. P. C. R. This is reverse transcription PcR. Um And you this will is a special technique using special dyes but essentially the same process of these multiple cycles. Um To say this is how much D. N. A. Is in this sample and this is how much D. N. A. Is in all these samples. So it's super important. So this is an example. So we start out with some D. N. A. Fragment that we want to amplify here in red. So we take primers essentially and they bind here. They bind here and they say D. N. A. Plum race come bind the D. N. A. Plum race will come it'll bind there and it'll replicate. It'll come it'll bind here and it'll replicate. And so what you get is that after cycle one you get four copies after cycle to you get a copies. I don't have cycles here. But if I did it would be 2468 10 12 14 16 copies. Um And so on so forth. And like I said this is 25 to 40 X. Cycles. So you can imagine the number of DNA molecules you get is super super a lot. I think that can be used in a laboratory setting, so that's pcR let's not move on.
2
Problem
Problem
How many copies of DNA exist after 4 cycles of PCR?
A
4
B
8
C
16
D
32
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Problem
Problem
Which of the following shows the correct order of steps for PCR?
A
Melting DNA strands → Primer Binding → DNA polymerase replication
B
Primer Binding → Melting DNA strands → DNA polymerase replication
C
Melting DNA strands → DNA polymerase replication → Primer Binding