72 Gel Electrophoresis and DNA Fingerprinting
Dr. Lisa Bartee and Jack Brook
Gel Electrophoresis
DNA is a very negatively charged molecule because each phosphate group in each nucleotide has a negative charge (Figure 1). This means that if an electric current is run through a DNA sample, the DNA molecules will move towards the positive charge of the current. Scientists take advantage of this property of DNA in order to separate DNA molecules by size.
Agarose is a molecule that is purified from seaweed. Agarose powder is mixed with water, then heated until the powder dissolves. The mixture is poured into a tray (Figure 2) and allowed to cool. As it cools, it forms a semi-solid gel. This process is similar to making Jell-o, if you’ve ever done that. This gel contains microscopic pores (holes) of different sizes. You can imagine the gel structure as looking like a thick hedge or blackberry bush. The structure of a hedge or blackberry bush is similar to the polysaccharide matrix that makes up an agarose gel (many molecules criss-crossed every which way).
DNA can be drawn through the agarose gel using an electric current because the negatively charged DNA molecules are attracted to a positively charged electrode. Small DNA molecules (short nucleotide chains made of a small number of base pairs) are able to move relatively rapidly through the gelatinous agarose matrix. In contrast, large DNA molecules (long nucleotide chains made of a large number of base pairs) move much more slowly. You can compare the movement of DNA through an agarose gel to the movement of animals of different sizes (representing the DNA) through the thick hedge or blackberry bush (representing the structure of the agarose gel). Small animals, such as rabbits, can move quickly through a blackberry bush just like short DNA molecules can move quickly through the agarose gel. Medium-sized animals, such as German Shepherds, move much more slowly than rabbits. Large animals, such as cows, wouldn’t be able to move very quickly through a blackberry bush at all. We can use this separation of DNA molecules by size to determine how large DNA molecules are within a sample. We can also compare the sizes of DNA molecules within several different samples.
One individual DNA molecule cannot be seen on a gel. However, all of the DNA molecules that are the same size will move the same distance through the gel. They will form a “band” of DNA that can be seen (Figure 4). The intensity or darkness of a band is due to the number of molecules of DNA that are running at that position on the gel. More DNA will cause the band to be darker. Less DNA will cause the band to be lighter.
The size of DNA molecules is usually measured in base pairs (bp). One base pair consists of the nucleotides on the two strands of DNA that are hydrogen bonded together. For example, if there is an A on one strand of the DNA double helix, there would be a T on the second strand. The A and the T together are referred to as one b