How Biologists Separate Molecules with Gel Electrophoresis

Scientists use gel electrophoresis to separate molecules based on their size and electrical charge. Gel electrophoresis can separate fragments of DNA that were cut with restriction enzymes, creating a visual map of fragment size that’s easy to interpret. Or scientists may use gel electrophoresis to separate a protein they want to study from other proteins in a cell.

One of the advantages of gel electrophoresis is that scientists can separate several samples side by side so they can compare them. The comparison of separated DNA molecules is the basic method behind the DNA fingerprints that forensic scientists use to compare samples from crime scenes with those of suspects.

Scientists conduct gel electrophoresis by inserting molecules such as DNA into little pockets called wells within a slab of gel. They then place the gel in a box called an electrophoresis chamber that’s filled with a salty, electricity-conducting buffer solution.

The DNA molecules, which have a negative charge, move toward the gel box’s positive electrode because opposite charges attract. When the scientists run an electrical current through the gel, the gel becomes like a racetrack for the DNA molecules as they try to get to the positively charged end of the box.

When the power is turned off, all the DNA molecules stop where they are in the gel, and the scientists stain them. The stain sticks to the DNA, creating stripes called bands. Each band represents a collection of DNA molecules that are the same size and stopped in the same place in the gel.

In order to work with the information from the gel more easily, scientists can make an identical copy of the gel by transferring the DNA molecules to a thin sheet of nylon or nitrocellulose, a strong but flexible material that binds to DNA. This procedure is called making a blot of the gel. A blot on a thin, flexible material can be handled, whereas the original slab of gel can crack and break.

1. The following figure shows a linear piece of viral DNA that’s 27 kb long and has restriction sites for the enzymes A, B, and C. After the viral DNA was cut with various combinations of restriction enzymes, the resulting DNA fragments were separated using gel electrophoresis.

   Based on the information given in the figure, what pattern of bands would you predict in the lane of the gel marked A + B, which would be loaded with many copies of viral DNA cut with both enzymes A and B?

   

2. If you treated the viral DNA with all three restriction enzymes — A, B, and C — and then separated the fragments using gel electrophoresis, what pattern of bands would appear in the final lane of the figure?

   1. Bands would appear at the positions indicating 3, 5, 8, and 11 kb

   2. Bands would appear at the positions indicating 4, 8, and 15 kb

   3. Bands would appear at the positions indicating 3, 7, 8, and 9 kb

   4. Bands would appear at the positions indicating 3, 4, 5, 7, and 8 kb

3. The open rectangles at the top of the gel in the figure represent the wells. Towards which end of the gel would the positive electrode be located?

   1. The top

   2. The bottom

4. The open rectangles at the top of the gel in the figure represent the wells. If you were to run a sample of uncut viral DNA through this gel for the same amount of time that the other samples were run, at which end of the gel would you expect to find your band?

   1. The top, near the wells

   2. The bottom, away from the wells

The following are the answers to the practice questions.

1. The viral DNA has a restriction site for enzyme A at a position 8 kb from the left end of the DNA. So, cutting with enzyme A will generate some fragments that are 8 kb in length.

   The viral DNA also has a restriction site for enzyme B at a position just 4 kb from the restriction site for enzyme A, so cutting with restriction enzyme B will produce some fragments that are 4 kb in length. The cut with enzyme B will also produce remainder fragments that stretch from the restriction site for B all the way to the right end of the viral DNA. These remainder fragments will be 15 kb in length.

   To double-check your work, you can add up the lengths of your predicted fragments: 8 + 4 + 15 = 27, which is the correct length of the entire piece of viral DNA. To determine the position of the bands on the gel, look at the size labels along the right side of the gel. You would predict a band of DNA fragments to appear level with the 4 kb marker, the 8 kb marker, and the 15 kb marker. All these bands should contain the same numbers of fragments so they should appear in equal thickness on the gel.

2. The answer is 4. Bands would appear at the positions indicating 3, 4, 5, 7, and 8 kb.

   If you cut the viral DNA with all three enzymes, you’ll make four cuts, resulting in five fragments.

3. The answer is 2. The bottom.

   DNA, which is negatively charged, is loaded into the wells. The positive electrode is located at the far end, away from the DNA, so that it will attract the DNA from a distance, pulling it through the gel.

4. The answer is 1. The top, near the wells.

   Uncut pieces of viral DNA would be longer than any of the fragments made by restriction enzymes (they’d be the entire 27 kb long), so they would travel a shorter distance than any of the fragments.