18. Molecular Genetic Tools
Methods for Analyzing DNA
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Methods for Analyzing DNA and RNA
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Hi this video we're gonna be talking about methods to analyze D. N. A. And R. N. A. So this is the methods video. So of course we're gonna be talking about different techniques that geneticists used to study DNA and RNA. So one type of technique is called a library and libraries are collections of bacterial clones. So this is a bacteria that has been grown and produces the same type of D. N. A. Which is a clone but it has a ton of different bacteria in it so that we can actually see it and extract it visualize it. They all contain the same D. N. A. And there's multiple of them. Right? So collection so you have hundreds thousands of different bacterial clones and they each contain a different genetic sequence. Now the type of sequence that it contains depends on how the library was created. So you can have genomic sequences which are gonna be small fragments of the genomic D. N. A. And you also can have C. D. N. A sequences. See DNA libraries that are made from complementary D. N. A. Now why would we need both of these? What do these different things tell us? Right. Well the genomic D. N. A. Is giving us information about the genomic sequence. Right. And the genomic sequence contains a lot of information that isn't necessarily coding for proteins. Right. This contains regulatory regions it contains enTR ONs it contains non coding are N. A. S. Right? And all these things are found in genomic libraries when we use the complementary DNA or the C. DNA libraries. This complimentary D. N. A. Is made directly from M. RNA. Remember complimentary D. N. A. Is reverse transcribed from the M. R. N. A. Into the complementary DNA. So you start with the RNA. Of the organism and you turn that into complementary DNA. Well if you're starting with the R. N. A. You've already gotten rid of the N. Tron you've gotten rid of the non coding regions. You've gotten rid of the regulatory regions. And so the complementary DNA libraries represents what the protein coding region is. What is that sequence. And so see DNA libraries are super important to look at you know what proteins are being expressed at that certain time. And genomic libraries are good at looking at the entire genome which includes a lot of things that are not protein coding. So that's why we have the two different ones. So how you essentially get a genomic libraries. You have an organism with some D. N. A. It could be a cell, it could be a whole bacterium. Whatever you want to do. You extract that D. N. A. And you get it out in a laboratory setting, you digest it with restriction enzymes. So now it's in a bunch of different fragments and then each of these fragments are put into cloning vectors. This is exactly like the cloning video that is in the other cloning video that we talked about. Put it into vectors, put it into bacteria and then each one of these is going to be a clone representing each one of these fragments. So there's going to be especially if you think of genomic libraries, how big the genome is that can be thousands, hundreds of thousands of bacterial clones that exist. So it's a huge amount of clones representing the DNA found in the genomic library or the C. D. N. A library. A second after that I want to talk about is blotting blotting is super important to separate and visualize and obtain actually DNA or RNA. So there's two different types of more than this. But it's too that we're going to talk about for DNA and RNA. So southern blots are going to separate the genomic DNA and then you have northern blots that are separating RNA sequences. So what this is essentially looks like as you take a DNA or an RNA sequence whatever you have multiple samples of it and likely they're going to be different sizes. So if we have to be a and this contains like a five kill obey sequence. So 5000 base pairs and we have to be and it has to with 2000 base pairs and 3000 this even though the total of the sequence is five right because they're different sizes, that's going to separate them differently. So we run them on these blots, essentially how these blocks are run is you take some kind of jelly like material almost like jello essentially um it's called agar. Which you may use if you're really into cooking. But you run these things on these gels and how they run is they generally run by charge because D. N. A. And R. N. A. R. Charged molecules. And so if you put a positive at one end and a negative the other D. N. A. Is negatively charged, it's gonna move towards that positive. So the first thing is you have this gel with a negative end and a positive end and you input your D. N. A. Or your R. N. A. In here. And because DNA is negatively charged it wants to move towards the positive now because it's like jello material right? That's going to mean that it has all of these, it's kind of thick, it's hard to get through. So the bigger you are the less far you're gonna be able to travel. So the bigger the D. N. A. Sequence, the less far you're gonna be able to travel. Which is why when you get five killer bases it's up here because it hasn't traveled very far, right? Whereas if you get your three Kb and your two Kb pieces, these are going to travel at much lower sizes. And we run these things called ladders that have bands at each one of these. So we know exactly what size it is. Um And so you run these things on a gel and then usually for blotting it's called blotting because you actually have to blot it. Um and so you have to transfer this onto some kind of membrane which they again do through charges. But instead of having negative and positive here, if this is your thing and this is front and back, we do negative and positive this way where it will run that way onto a membrane and you don't necessarily like you're not going to be quizzed on these steps that I am going over because you may actually do them in a laboratory setting. And so I just want to sort of refresh your memory for this. So you transfer this onto a membrane. And then third, the reason we call it blotting is because you bought and um you can block with different things. There are different chemicals that recognize D. N. A sum that recognize our N. A. There are even some that I'm not talking about here but there are some that recognize proteins and you take this membrane and you put it with those DNA or RNA probes or different chemicals that recognize DNA. RNA. Let it sit for a while, then you wash it off. And that's what it looks like because these probes have bound to the regions with the D. N. A. Or RNA in them or protein. But in this case we're talking about DNA or RNA. So like I said, I didn't write the steps because this isn't allowed class. Um but you're probably going to be doing these in a lab setting if not in this class, definitely in another. And so just wanted to refresh your memory on how these things work. But this part you won't be tested on this part. You will so know what the different plots are and what they do and how they separate out the D. N. A. By size. And so with that let's not move on.
2
Problem
ProblemIn terms of molecular biology, what is a library?
A
A place where molecular biology textbooks are stored
B
A collection of clones containing short genetic sequences
C
A collection of clones containing a large collection of proteins
D
A collection of clones with different mutant phenotypes
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Problem
ProblemIf you had a sample of RNA to analyze, which of the following techniques would you most likely use?
A
Southern Blots
B
SDS-PAGE Blots
C
Northern Blots
D
Western Blots
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