A technique used to separate DNA fragments and other macromolecules by size and charge.

Key points:

  • Gel electrophoresis is a technique used to separate DNA fragments according to their size.
  • DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel.
  • DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
  • When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.

Introduction

Suppose you have just done a PCR reaction, making many copies of a target DNA region. Or perhaps you’ve done some DNA cloning, trying to "paste" a gene into a circular DNA plasmid.
Now, you want to check and see whether your PCR worked, or whether your plasmid has the right gene in it. What technique can you use to visualize (directly observe) the fragments of DNA?

Gel electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another.
The suffix phoresis means "migration" or "movement." 1^1 The prefix electro tells us that we are using electricity to make molecules migrate.
All DNA molecules have the same amount of charge per mass. Because of this, gel electrophoresis of DNA fragments separates them based on size only. Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another. We can also determine the absolute size of a piece of DNA by examining it next to a standard "yardstick" made up of DNA fragments of known sizes.
Separation of molecules in electrophoresis is based on charge (the thing that gets pulled) and the effective cross-section of the molecule in whatever state it finds itself – folded, unfolded, bound to other molecules, etc.
A collection of DNA fragments separate by length because they are all the same type of molecule. In general, the only meaningful difference between the various fragments should be their length.
However, there are some exceptions to this rule. For instance, some DNA molecules are circular (like bacterial plasmids), while others are linear. Circular DNA molecules may run differently than linear ones through a gel. Plasmids, for example, can exist in a form called "supercoiled," in which they actually move faster through a gel than they should for their size, because they have twisted into a skinny shape that can move through the gel more easily.

What is a gel?

As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.
At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed:
Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current. Although you may not be able to see in the image above (thanks to my amazing artistic skills), the buffer fills the gel box to a level where it just barely covers the gel.
The end of the gel with the wells is positioned towards the negative electrode. The end without wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.

How do DNA fragments move through the gel?

Once the gel is in the box, each of the DNA samples we want to examine (for instance, each PCR reaction or each restriction-digested plasmid) is carefully transferred into one of the wells. One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments.
Next, the power to the gel box is turned on, and current begins to flow through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they start moving through the matrix of the gel towards the positive pole. When the power is turned on and current is passing through the gel, the gel is said to be running.
A typical voltage for running an agarose DNA gel would be in the range of 8080 120 V120 \text{ V}. A higher voltage will make the gel run faster, but may also melt it if it runs for a long period of time. A lower voltage will make the gel run more slowly (which can be convenient if you want it to finish up at a particular time, say, after you get back from lunch).
DNA samples are loaded into wells at negative electrode end of gel.
Power is turned on and DNA fragments migrate through gel (towards the positive electrode).
After the gel has run, the fragments are separated by size. The largest fragments are near the top of the gel (negative electrode, where they began), and the smallest fragments are near the bottom (positive electrode).
Based on similar diagram in Reece et al.2^2
As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for awhile, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells. Very short pieces of DNA may have run right off the end of the gel if we left it on for too long (something I've most definitely been guilty of!).

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel.
The bp next to each number in the ladder indicates how many base pairs long the DNA fragment is.
A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. A single DNA fragment (or even a small group of DNA fragments) would not be visible by itself on a gel.
By comparing the bands in a sample to the DNA ladder, we can determine their approximate sizes. For instance, the bright band on the gel above is roughly 700700 base pairs (bp) in size.

Check your understanding

Leftmost lane: ladder with 3000 bp, 1500 bp, and 500 bp bands marked on it.
Lane 1: 5000 bp band.
Lane 2: 100 bp band.
Lane 3: 1500 bp and 2000 bp bands.
Lane 4: 500 bp band.
Four lanes are numbered on the gel above. (A lane is a corridor through which DNA passes as it leaves a well.)
Which lane matches each description below?
 
11
22
33
44
This lane contains the longest DNA fragment.
This lane contains the shortest DNA fragment.
This lane contains a 15001500 base pair (bp) DNA fragment.
In a DNA gel, the longest DNA fragments migrate most slowly through the gel and stay closest to the wells (where the are initially loaded into the gel). The shortest DNA fragments migrate most quickly through the gel and get the closest to its far end (away from the wells).
Thus, the band representing the longest fragment will be closest to the top of the gel. This band is found in lane 11.
Similarly, the band representing the shortest fragment will be closest to the bottom of the gel. This band is found in lane 22.
We can use the DNA ladder (in the leftmost lane) to figure out which lane contains a 15001500 bp band. If we trace the 15001500 bp band of the ladder rightwards across the other lanes, we find a band of matching size in lane 33.
 
11
22
33
44
This lane contains the longest DNA fragment.
This lane contains the shortest DNA fragment.
This lane contains a 15001500 base pair (bp) DNA fragment.

Attribution:

This article is a modified derivative of:
The modified article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

  1. Phoresis. (2011, April 14). Retrieved May 31, 2015 from Wikipedia: https://en.wikipedia.org/wiki/Phoresis.
  2. Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey, J. L. (2012). Figure 12.13. Gel electrophoresis of DNA. In Campbell biology: Concepts & connections (7th ed., p. 243).

References:

Agarose. (2015, January 19). Retrieved April 3, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Agarose.
Gel electrophoresis. (2014). In Scitable. Retrieved from http://www.nature.com/scitable/definition/gel-electrophoresis-286.
Kratz, R. F. and Siegfried, D. R. (2010). Using gel electrophoresis to separate molecules. In Biology for dummies (2nd ed., pp. 132-133). Hoboken, NJ: John Wiley & Sons.
Oswald, N. (2008, August 6). Agarose gels do not polymerize! In Bitesize Bio. Retrieved from http://bitesizebio.com/10248/agarose-gels-do-not-polymerise/.
Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey, J. L. (2012). Gel electrophoresis sorts DNA molecules by size. In Campbell biology: Concepts & connections (7th ed., p. 243).
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Using restriction enzymes to make a recombinant DNA plasmid. In Campbell biology (10th ed., pp. 413-414). San Francisco, CA: Pearson.
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