Biology: Southern Blotting

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If you've gotten the feeling from just thinking and reading and listening about molecular biology and biotechnology that one thing builds on the other. If that's the idea that I've given you, that's good, because it does. And you know, there's certainly an unbelievable wealth of material out there being developed every day that is new. And, everyday, a research scientist has to literally keep in tune with the papers that are being presented because new techniques - not every technique is going to win a Nobel Prize, and sometimes these things kind of like, stay hidden until you need it. When you say to yourself, "Man, I need a way, other than putting it in a gel, I need a way to really make my probe stand out because how do I get this radioactive probe into the gel?" Imagine this, as an example. You've got this gel, that you just ran, a DNA gel, and you put all your DNA in there and you know if you've been reading, we kind of slide it, and you say "Oh, yeah" and then you probe it. Then you add a piece of single - first you make this single strand. Well, how do you do that? And then once you make this DNA in that gelatinous matrix, if you make that single stranded, and I say, "Ah, just add a probe, add a single-stranded probe." Well, what do you do? Sprinkle it on there? The stuff is in the gel. And so the next logical step that we had to solve is, how do you get stuff out of a gel so that you can mess with it. Now one way to get stuff out of a gel was very simple. If I just want to work with this particular piece of DNA, I use a very sophisticated tool called a razor blade, I cut that little piece of auger, because it's just gel, it's augerous. I cut that little piece of augerous out and I take that DNA and I put it in a test tube and I dissolve the augerous. And I end up with pure DNA. That's the way to purify a gene, or a piece of DNA.
That's nice, but aren't there ways to get - suppose I want to keep this thing in its entirety and I don't want to cut chunks out of there. Well, one more technique I want to tell you about. It was developed by a guy whose name was Southern and we call it the Southern blot. And the Southern blot is probably the state of the art way to visualize probes and markers. Visualizing doesn't mean "I'm visualizing...", like we normally use it. Visualize means to make it stand out, to develop it, if you will. So let me take you through - what seems so low tech to some first time researchers, and yet is such a powerful technique. How does a Southern blot work? Very simply. Remember that DNA? Say I have my DNA on a gel and I have all these bands on here and I want to probe this. I want to somehow know if any of this DNA contains the following sequence, AAATTT. I want to know that. How am I going to know that? Well, you already know about DNA hybridization. If I could get this to hybridize to this in its single-strand form, then I'll be cool, right? If this will hybridize with any of this DNA, I'll know it's there.
Well, that's easier said than done. Because, how am I going to get the DNA out of the gel? Southern blotting. Here's what you do. You take that gel and you put it in a tub - glass is fine - and you put a little platform there, like so, a little plastic platform. And you take that gel and you put it on there. But first, you fill this tub with a liquid and in that liquid will be a variety of buffers and chemicals whose job it is to denature the DNA. So, it's going to be a chemical treatment now. And, what you do is you put on that a piece of filter paper. On that platform, we're going to put a piece of filter paper, like so. And on top of that, we're going to put our gel with our DNA. So, we're looking at that from the side, you get it? We're looking at it from the side. And here comes the key ingredient folks. On top of the gel, we put a piece of paper, but it's a special paper and that paper is positively charged. It's a nylon membrane and it can contains a positive charge. Now, why is that so important? What's the charge on DNA? Yep, negative. So, if the paper is positively charged, what's going to happen? Well, if somehow this liquid, if we can get this liquid to soak up this blotter, and then somehow soak up into this membrane, then the DNA will stick to the membrane. So what are we going to do? You wouldn't believe how simple it is.
On top of this paper, we're going to stack some more filter paper. And then on top of that filter paper, we're going to put paper towels, stacks of paper towels so that if it was on this table, I would have this thing stacked about this high. And then on top of that, we put anything we can find that's heavy. We put big bottles full of liquid. We put metal weights. We put books. Anything that's heavy, because look what's going to happen. Like paper towels are supposed to, the liquid is going to soak up out of here; it's going to pass through the gel, it's going to denature the DNA on the way to the gel; it's going to leave the DNA on here while the liquid continues to soak up through what we call the wicking system. You come back the next night, these paper towels are soaked, and what you have is a piece of nylon membrane that, theoretically - I smile because I've had nylon membranes that only theoretically had DNA had in them. Well then what? Then you use the next sophisticated device called a baggie. A plastic bag. You put that gel in that plastic bag and you add your probe. Remember the probe? I don't even remember the probe I made up, but let's just say it was AAATTT, radioactively labeled. So think about it. Do I know have single stranded DNA on that nylon membrane in that bag? I surely do. Because the bag has that membrane in it and that nylon membrane, even though you can't see it because DNA is clear and this is not stained, has those bands. So wherever this probe lands, what's going to happen? Wherever the probe lands, you're going to get that DNA hot. And then when we're done, we take this off; we wash it which washes off all of the excess probe. Of course, the DNA stays in there because it is negatively charged and then we do an autoradiogram, ARG. In other words, we develop a photographic plate. What's our plate going to look like? It's going to look like a negative. But guess what? We will see a band there and a band there when it's developed, showing that this probe indeed did hybridize in two places. Southern blotting is the way to finally demonstrate whether a gene you're looking for as long as you have that probe. Whether the gene you're looking for is indeed part of the DNA you digested.
If you just think about it, there's an enormous number of things you can use this for. Even if you think back to VNTR's and fragment linked polymorphisms, even you can put those from a gel onto a Southern blot. Very, very useful technique. Southern blotting. It's what a lot of you will be using one of these days.
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