The explosion of molecular biology techniques that began in the mid-1970s (and continues today) has provided tools to examine the physical structure of DNA, its nucleotide sequence and how genes are read and regulated. One key tool is the ability to visualize DNA molecules and determine their length by using a technique called gel electrophoresis. Introduction to gel electrophoresis
In gel electrophoresis, DNA fragments move through a porous matrix made of agarose, a gelatin-like substance purified from seaweed. The agarose is melted like Jell-O and then poured into a plastic tray to harden into a slab called a gel.
A plastic comb inserted at one end while the gel is hardening forms wells where DNA samples can be placed. The DNA is mixed with a loading buffer that contains glycerol—this makes it heavier than water, so it will sink to the bottom of the well. The gel is then covered with a buffer solution that can carry electric current, and electrodes are placed at each end of the gel and connected to a power supply.
Because DNA is negatively charged (each nucleotide has a negatively charged phosphate attached to it), it will move toward the positive electrode.
Larger molecules move through the agarose more slowly, while smaller ones can slip through the pores faster. So, the fragments wind up arranged in order according to size, with the smaller ones having moved farther toward the positive pole. Figure 47 shows an example. Because the DNA is invisible, the loading buf fer also contains two dy e s : bromophenol blue (a small dye molecule that behaves like a DNA fragment about 600 bases long) and xylene cyanol (a larger dye that acts like a DNA fragment of about 4000 bases). These dyes form lines that give you an idea of how far your DNA has moved. Some loading buffers also have a third dye, behaving like a very small DNA molecule (50 bases or so).
As the DNA migrates, the different fragments will form bands; each band is composed of many identical copies of a particular-size piece of DNA (you can’t do gel electrophoresis with one DNA molecule: you need millions or billions of identical molecules). The last step is to make the DNA bands visible, using a fluorescent molecule that inserts between the bases in the DNA helix. We use a commercial loading buffer called EZ-Vision which includes the fluorescent molecule, so the gel is already stained when it’s done running. Another method is to soak the gel in ethidium bromide after running it. Either way, the bands can be seen using ultraviolet light and photographed to make a permanent record.
Of course, gel electrophoresis requires some kind of DNA sample—a plasmid, a PCR product, a segment of a chromosome, etc. If the molecule is circular, enzymes are used to cut the DNA (see the section on restriction digestion, page 87), because circular molecules can be either tightly or loosely coiled and don’t wind up at the same place on a gel as a linear molecule of the same size. Whatever your sample is, it must be mixed with loading buffer (containing glycerol and dyes, as described above) before electrophoresis. Add a volume of loading buffer equal to 1/5 the volume of your sample and mix it well before loa