Comparing DNA

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Lebanon Valley College *

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MISC

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Biology

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Nov 24, 2024

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Section Objectives  Students will be able to describe a technique used to compare DNA samples. Introduction One important technique used in the laboratory is gel electrophoresis . Gel electro-for- what? Gel electrophoresis is a long name for a simple concept: comparing the length of two sections of DNA. Why would you want to compare the length of one section of DNA to another? It turns out that this is one of the fastest methods for isolating a section of DNA that you are interested in studying. Setting up Your Experiment Say you’re a biologist studying a new gene, NLB -1. Using information from the Human Genome Project, you’ve seen that the gene is 10,000 DNA bases long. By looking carefully at the DNA sequence, you’re able to see that there happens to be an EcoRI restriction site on each end of the gene. This means that if you mix a DNA sample with EcoRI, it will cut out the NLB-1 gene from the rest of the DNA. There’s a major problems with this. Can you think of what they might be? It has to do with EcoRI. EcoRI will not only cut out your gene. There are bound to be many other EcoRI sites somewhere in the three billion bases of the human genome. So when you treat your DNA sample with EcoRI, it is going to cut it up into who knows how many pieces. How do you find the NLB- 1 gene in all this mess? You can’t see the DNA, you can’t read the DNA sequence to find the gene. What’s next? You have one very important piece of information about NLB-1: its size. You know that it is 10,000 base pairs long. It’s just like looking for a friend in a crowd: you can’t look at all their faces, so you scan for people of about the right height as your friend. The key to finding NLB-1 is to separate the DNA out by its size. DNA Gels Biologists discovered that there is a certain kind of gel, called acrylamide, that DNA will move through if there is an electrical current pulling it (The word “acrylamide” is similar to the word “acrylic,” like acrylic fabrics or paints). Electrical current can pass through this gel. As it does, DNA gets pulled from one side to the other.

Separating DNA Just because DNA can be pulled along by electricity doesn’t tell us how to separate NLB - 1. But, just like cars, smaller DNA moves faster through the gel than larger fragments. If you put DNA in one end of the gel, the smallest fragment will get to the far end first, and the largest fragment will still be near the starting line. DNA gels are always organized into rows. In one row you put your DNA sample that has been treated with EcoRI. Somewhere floating around in the sample is NLB-1. In another row you add a set of standards . The standards are made of up fragments of DNA that you already know the length off. You then plug in the gel and let it run for some time. Next, the gel is treated with a special stain that binds to DNA. Viewed under ultra-violet light, the stain glows and you can see all of your DNA sample. The standards have special dies that glow different colors depending on their length. Each chunk of DNA shows up as a separate band, as in Figure 1. Figure 1: A DNA gel with standards. The standards are on the left and four different DNA samples are in the rows on the right. The band near the bottom is the smallest and has traveled the furthest because of the electrical current. How do you find NLB-1? You first look for the standard that is 10,000 bp long. Then you look for a band in your DNA sample that is the same length. In order to isolate your sample, all you have to do is get a small knife and carefully cut out the band. You can then dissolve the gel in a test tube, giving you a pure sample of just your gene. This will allow you to do other experiments with the gene, like put it into a plasmid, clone it, or even prepare it for gene therapy. Summary If you want to isolate a single gene you can use restriction enzymes and DNA gels. After you cut up your DNA sample with restriction enzymes you can load it into a gel along with a set of standards. You then run an electrical current through the gel. By comparing the standards to your sample to the length of the DNA, you can locate it on the gel. You can then cut the band out from the gel and dissolve the gel in a test tube.

Concept Reinforcement 1. Here is a gel with a set of standards and a DNA sample. The red standards it 1,000 DNA bases long, the orange 2,000, the green 4,000, and the purple 5,000. Which one of the DNA sample bands is 2,500 DNA bases pairs long? 2. Why is important to include standard when you run your DNA sample on a gel? 3. Explain why size is the best way to find a specific piece of DNA once you have cut up a sample with a restriction enzyme. 1000 5000 Standard DNA Sample

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