Gel Electrophoresis

Gel Electrophoresis


DNA carries a negative charge on the phosphates all along the backbone. As a result, if you put it in an electric field, it will migrate towards the positive pole. Since the charge goes up with the size (each base pair adds more negative charges), all pieces of DNA would move at the same rate in a fluid.
However, if you make it move through a gel…something with small-ish pores such that the DNA has to wind it's way through, bigger pieces of DNA get slowed down more than smaller pieces, and you can separate them by size.

Often we use a gel called "Agarose," based on agar from kelp. It's a clear-ish gel that can be made with a buffer that conducts electricity.

The other tool we need is a way to cut DNA into predicable sizes. For that, we use something called "restriction enzymes." These are sort of the innate immunity of bacterial cells. They recognize foreign DNA and cut it at specific sequences.

Screen Shot 2018-05-11 at 9.13.51 AM


Above is a drawing of a "plasmid" like the one we used in an experiment earlier. It can replicate in bacteria and has the gene for resistance to ampicillin. I've highlighted the cut-sites for an enzyme called EcoRI (Eco is for E. coli, the bacterium from which it was isolated, and RI just is the isolate number, when it was first found).
The protein binds to DNA, seeks out the sequence GAATTC, and then cuts between the G and the A on each strand.

There are a total of three occurrences of GAATTC in this particular plasmid, so treating this DNA with EcoRI would cut it into 3 pieces. The whole plasmid is itself almost 15,000 base pairs (or 15Kbp for "kilo base pairs").
Suppose I had 108 pieces of this circular DNA and cut, or "digested," it with EcoRI. I would get 108 pieces of DNA of each of the three sizes. If I loaded them into a gel and ran them in an electric field, the pieces would separate based on size.

Below is a simulation of what the gel might look like:
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The DNA is loaded into the little wells at the top, then the current is turned on for 30 minutes or so. The first "lane" is a molecular weight marker, which has pieces of DNA of known sizes. I can use that to see how big the other pieces of DNA are. Notice that the smallest piece, highlighted in blue in the lower left panel, is 1,401 base pairs (bp) long. You can see on the circular map where the highlighted piece comes from. The simulated gel, upper left, shows that the highlighted band of DNA runs just below 1,500 bp fragment in the marker lane.
The middle band is predicted to be 2,712 base pairs and it runs just below the 3000 base pair fragment in the marker lane. The largest piece is 10,796 base pairs, and it runs, as expected, up with the largest piece in the marker lane.

What is this used for?



The most common way we use is to verify that we have the right plasmid DNA cloned. I might compare some DNA I purified to the expected pattern above to make sure it really was the right plasmid.
Back in the 1980s and 90s, there was a popular technique for comparing human DNA, say that found at a crime scene or to establish who someone's father is. You could cut the suspected father and child's DNA with an enzyme and run it on a gel. There is so much DNA in a human that it would just be a continuous smear of DNA. But, there are tricks to use a radioactive DNA probe for, let's say the cytochrome C gene, and therefore look at the sizes of DNA from that gene only. You might then compare to see if the suspected father has a similar pattern to that seen in the child, or if DNA from the suspected criminal matched DNA found at a crime scene.
It's not used much anymore…but I've seen it in prep books.

Gene cloning


Finally, the restriction enzymes are used for gene cloning. Since EcoRI always cuts at GAATTC (between the G and the A on each strand), any DNA cut with it leaves a single stranded overhang of AATT. We call that the "sticky end" or "cohesive" end. Two pieces of DNA cut with that enzyme can stick to each other because of the hydrogen bonds between the As and Ts. We then use Ligase (remember, it splices together to ends of DNA during DNA replication and removal of Okazaki fragments).
That's how we splice two pieces of DNA together. We can build all kinds of interesting things that way.

Here is a video of some of your classmates making and using a gel.
It's not sized all that well. Just size it down and you should be able to view it fine. It's less than 5 minutes.