18. Molecular Genetic Tools
Genetic Cloning
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Genetic Cloning
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Hi in this video we're gonna be talking about genetic cloning. So the ability to clone genes is the basis for all modern genetic advancements. I mean the majority of things that we know about genetics today is due to the fact that we can clone genes and actually be able to study them 1x1. So I'm gonna talk to you about what genetic cloning is and the steps to do it. So essentially genetic cloning is just taking some kind of D. N. A. Of interest and putting it into a system where you can develop a lot of it to study it. So genetic cloning starts with amplifying the D. N. A. Of interest. So you have some gene that you want a lot of copies of. And so you amplify and there's many different ways to do this. But one of the most common and the one mentioned in your book is preliminaries chain reaction. So preliminary chain reaction consists of three steps. The first is that you heat the D. N. A. Strands to a very high temperature and that causes the double helix to separate into two single strands. Then you lower the temperature. And that allows for small nucleotide primers to a Neil. And that tells the preliminaries where it's gonna start. And in the third the preliminaries comes on binds those primer says here, I'm gonna start and that replicates the D. N. A. And you do this in multiple cycles over and over and over again to create your to create multiple copies of this DNA of interest. Now you can do this using genomic DNA. But you can also use it to create an amplify see DNA from RNA. Um So you take the RNA. You reverse transcribe it into C. D. N. A. And then that DNA can also be used to amplify through PCR. So here is generally what PCR starts with. You have your first copy through cycle one. You get to through cycles to you get four. And then each one of these creates two and it keeps going for as many cycles as you want. Typically Pcr just 20 to 35 cycles. So you can get millions and millions of copies of D. N. A. After these cycles. Like I said if you start with D. N. A. This is a double strand right? Because DNA is double stranded. In the first step you heat And that creates two single strands that are now separated right? They're not bound. Then in the second step we move away and may change the color to primers come in and a Neil stay here and here. And in the third step the polymerase binds the primer and amplifies, binds the primer and amplify. So then what you end up with is two strands like this. And then you repeat this process over and over and over again say 35 cycles. And you get a ton of DNA at the end. That's the first step. The second step to cloning is you take this D. N. A. That you've amplified and you have to cut it into small fragments. And the way to do that is through restriction enzymes. Which we've talked about previously restriction enzymes are proteins that chop D. N. A. It's very specific sequences. They create two types of ends, blunt ends and sticky ends. And these sticky ends are shown here. So if we have A D. N. A. Of interest we take a restriction enzyme which is here Echo R. One. It cuts right here. So this very specific sequence it cuts at that sequence. And so you can see when it cuts it creates two fragments. And these are sticky ends because their sequence overhang. So these four nucleotides to have nothing to bind to. And these four nucleotides have nothing to bind to. And that's called a sticky end. If it was if these weren't here and it was just this and this those would be blunt ends. Now, sticky ends, the creation of sticky ends is super important. And typically the way that cloning has happened. So if a blunt end occurs through that enzyme it just so happens to cut blunt ends. Usually there has to be some kind of next step to create sticky ends because we're going to see that sticky ends are going to be very important. So the next step is pacing the D. N. A. Into a vector or the word that you might be familiar with is a plasma. But essentially they're the same. And we call this whenever we take that DNA that we've amplified and not cut and we put it into a vector or plasma we call that were competent D. N. A. So how you do this is you have A. D. N. A. It has the sticky ends so it has some nucleotides here that are free to bind but they have nothing to bind to. And you use DNA ligations to bind those into a plasma. So sticky ends we usually make the sticky ends in the D. N. A. Itself, but also in the plasma. So you cut that plasmid with the same restriction enzyme. It creates the same sticky ends. And that way you can just paste it and paste that D. N. A. Straight into that plasma. So like I said before a vector used that term but that's just a bacterial plasmids or the sequence of interest this place there are many different types of vectors with different characteristics for different organisms etcetera etcetera etcetera. If you have really large. I mean like killer based upon killer base um fragments, DNA fragments especially if looking at genomic D. N. A. Um These are called bacterial artificial chromosomes or yeast artificial chromosomes. Here's the stand and these are the plasma is used for large inserts. Um And then for plant sales. A common one that's mentioned in your book is the T. One plasma. So essentially how this is done is you start out with D. N. A. And a restriction enzyme sort of cuts here and here. So now you have this with some sticky ends. Then you cut the same restriction inside with the vector and that gets pasted right into the vector. And then you can put the vector into a bacteria. Which is the next step here is that there were confident D. N. A. Is placed into bacteria. There's a few different ways to do this. Some involving heat, some involving electricity um some involving chemicals but essentially there's you get these D. N. A. Into a bacteria. Um You can also put it into a virus but typically it's done in bacteria. And that creates this transgenic bacteria that now has a gene that's not originally bacterial gene. So a trans gene is a new gene introduced into an organism. So for this case that's that red. And so now this bacterial is a transgenic organism because it contains a trans gene. And so we do this. Why do we do this? Why do we need to put this random gene into bacteria? Well bacterial bacterial grow rapidly right? So each time this bacteria divides which can be just essentially every few minutes it's going to create a copy of this um sequence. And it's going to grow it and grow and grow and it's also going to create that protein. So this is going to get expressed and it's going to create this protein version. And so we can isolate a ton of this vector or we can isolate the protein and use each one of these to study either the gene itself or the protein or any sort of the intermediates in between. And so this is why cloning is important because it allows us to create this protein to study or to use medicine or whatever we want to do with it. So that is genetic cloning. So now let's move on.
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Problem
ProblemThe purpose of polymerase chain reaction is to do what?
A
Create RNA templates
B
Create fluorescent probes
C
Amplify a short DNA sequence
D
Isolate proteins
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Problem
ProblemWhich of the following lists the steps of genetic cloning in the proper order?
A
PCR → Restriction Enzyme Cutting → Ligate into a Vector → Placed into Organism
B
Restriction Enzyme Cutting → PCR → Ligate into a Vector → Placed into Organism
C
Ligate into a Vector → PCR → Restriction Enzyme Cutting → Placed into Organism
D
PCR → Ligate into a Vector → Restriction Enzyme Cutting → Placed into Organism
Additional resources for Genetic Cloning
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