Gel Electrophoresis

Introduction

Congratulations on successfully performing a PCR! High five! Our samples are now fresh out of the thermocycler, and we have hopefully amplified our region of interest. That leads us to an important question: how can we see if the PCR worked? In other words, how can we visualize our PCR products? We’ll be utilizing a technique called gel electrophoresis coupled with a specific compound and conditions to help us visually analyze our PCR results. 

What is gel electrophoresis? Simply, this technique allows us to separate molecules based on their size and charge. Does anyone remember the electrical charge of DNA (figure 1)? Hint: think of that phosphate group! That’s correct, DNA is negatively charged! Because of this, we can exploit its characteristics to help separate our DNA pieces based on size and charge. 

Gel electrophoresis can be done with several different types of gels, one of the most popular and the one we’ll be using is agarose. This special type of gel is made from seaweed and is comprised of polysaccharides. It’s very flexible and for our lab, we typically order a container of agarose powder that we can combine with specific solutions to create a gel. What do these gels feel like? It’s like a tougher version of jello! When we’d like to perform gel electrophoresis, we simply weigh out a specific amount of agarose powder, add it to the solution, warm it up (usually in a microwave oven), and then pour it into a mold. This mold will have a comb that contains specific projections (see figure 2) to create holes through part of the gel called wells. This is where we’ll add our sample, in the wells. 

After allowing the gel to cool (depending on how hot the gel got and room temperature, anywhere between 15 minutes and 30 minutes), we’re ready to load our samples. First, we move the gel into a gel electrophoresis box and we add enough of the solution that we used to create the agarose gel to completely submerge the agarose gel. We are then ready to add our sample!

One thing we must add to the gel (there’s multiple steps in which we can add it; however, it’s easiest to do it right after pulling the molten gel out of the microwave) is something that will bind to our DNA and make it easy to visualize. There’s several different agents we can use for this, but the one we’ll be using has been deemed the “safest” for humans. The traditional substance used to visualize DNA is ethidium bromide which is an intercalating agent (meaning it wedges itself between the bases of DNA). One big downside to this agent is the fact it's a potent carcinogen! If one were to get intercalating agent in their DNA it would bind between bases and cause mutations potentially leading to cancer. So why was ethidium bromide even used? Well once it’s wedged itself between the bases of DNA in an agarose gel and the gel is exposed to UV light, the ethidium bromide will fluoresce, giving off light that we can see and allow us to visualize our PCR products as distinct bands in the agarose gel. 

Luckily, there’s much safer alternatives now, like Syber Safe the substance we’ll use to visualize our PCR products. This substance is simply added to the molten gel and it’s good to go!

So, we now have our agarose gel that has cooled in the gel electrophoresis box and we’ve added the solution so things are ready to go. What next? Now we load our samples into the gel. Our samples must contain loading buffer that helps impart a color (so we can see them running on the gel, to know when the gel has finished running) and glycerol that makes the samples denser than the solution in the electrophoresis box so that the samples will not float out of the wells. It’s important to take note of the order in which we load our samples into the wells. We want to know which sample is in which well, often it helps to write this loading order down so we can reference it later when analyzing our results. Typically, along with our samples, we’ll want to also load and run a DNA ladder or marker. This is just a predefined set of DNA fragments that will run at specific lengths. The manufacturer includes a guide that helps us figure out the size of our samples visually from the gel that we’re analyzing. 

Now it’s time to add an electrical current! Remember the charge of DNA? It’s negative! We know that if an electrical current is applied to our gel, the DNA will migrate toward the positive side of the gel electrophoresis box (we have positive and negative leads) because substances will migrate toward their opposite charge (positive will migrate toward negative, negative toward positive). But how will the DNA separate based on their size? The structure of the agarose gel is like a web (figure 3). As the electrical current is applied to the gel, the DNA will begin to migrate out of the well toward the positive side of the gel. The web structure will impede the longest pieces of DNA while the shortest pieces of DNA will migrate more easily through the matrix of the gel. After the loading dye markers have gone down the length of the gel, we turn off the electricity, and move our gel to a light box. There we can turn on the UV light and take a picture of our gel to then analyze our results. 

Overall, the smaller DNA pieces will migrate faster and end up closer to the bottom of the gel (opposite our wells) and the longest DNA pieces will migrate slowly and stay closer to the top of the gel (close to the gel wells).

Analyzing the Results

So, what do these bands in a gel mean? Generally, the stronger the intensity of the band, the more abundant the DNA. We typically find that shorter pieces of DNA are not as intense because they do not bind as much of the fluorescent dye, resulting in weaker appearing bands when analyzing the agarose gel under UV light (Figure 4). There are also various patterns of DNA banding that may appear that could indicate the structure of the DNA (for example, supercoiling of plasmid DNA has a very distinct pattern, Figure 5). 

We rely heavily on the DNA marker (or ladder) that was run in the gel. This allows us to compare the size of our DNA samples with known standards in the marker lane. There are numerous variables that could impact how the DNA samples run in the gel, so we must be aware that it’s not always set in stone, yet generally comparing our samples to the marker gives us an idea of the length of our DNA fragment and relative abundance (which can be extremely important if analyzing factors such as gene expression). 

Conclusions

Gel electrophoresis is an extremely useful technique for separating molecules based on size and charge. In our workshop sessions, we’ll be using agarose as the gel material for separating DNA amplified by PCR and later from restriction enzyme digests for the purpose of genotyping. Agarose is a polysaccharide that forms a complex web-like structure in a gel that allows us to separate out molecules based on their properties of size and charge. We focus on DNA but it’s important to know that other important molecules can be separated via gel electrophoresis. One of the most frequently used applications for gel electrophoresis is for the separation of proteins. Typically, this is done in another type of gel set up (a vertical running gel instead of horizontal) and a different material for the gel (polyacrylamide).

Overall, we’ve used gel electrophoresis to confirm that our PCR reactions have amplified a fragment of DNA of the correct size. We will use the remainder of the PCR reaction in a restriction enzyme digest that will allow us to distinguish wild-type DNA from mutant DNA (i.e. to genotype different individuals). Gel electrophoresis is a foundational technique that’s used everyday around the globe in multiple environments including science research labs and clinical diagnostic facilities.