Like the sequence-specific DNA-binding proteins we encountered in Chapter 7 (see Figure 7–8), restriction enzymes often work as dimers, and the DNA sequence they recognize and cleave is often symmetrical around a central point. Here, both strands of the DNA double helix are cut at specific points within the target sequence (orange). Some enzymes, such as HaeIII, cut straight across the double helix and leave two blunt-ended DNA molecules; with others, such as EcoRI and HindIII, the cuts on each strand are staggered. These staggered cuts generate “sticky ends”—short, single-stranded overhangs that help the cut DNA molecules join back together through complementary base-pairing. This rejoining of DNA molecules becomes important for DNA cloning, as we discuss below. Restriction nucleases are usually obtained from bacteria, and their names reflect their origins: for example, the enzyme EcoRI comes from Escherichia coli. There are currently hundreds of different restriction enzymes available; they can be ordered from companies that commercially produce them.
A variation of agarose-gel electrophoresis, called pulsed-field gel electrophoresis, makes it possible to separate extremely long DNA molecules, even those found in whole chromosomes. Ordinary gel electrophoresis fails to separate very large DNA molecules because the steady electric field stretches them out so that they travel end-first through the gel in snakelike configurations at a rate that is independent of their length. In pulsed-field gel electrophoresis, by contrast, the direction of the electric field changes periodically, which forces the molecules to reorient before continuing to move snakelike through the gel. This re-orientation takes much more time for larger molecules, so that longer molecules move more slowly than shorter ones. As a consequence, entire bacterial or yeast chromosomes separate into discrete bands in pulsed-field gels and so can be sorted and identified on the basis of their size (Figure 8–25D). Although a typical mammalian chromosome of 108 nucleotide pairs is still too long to be sorted even in this way, large segments of these chromosomes are readily separated and identified if the chromosomal DNA is first cut with a restriction nuclease selected to recognize sequences that occur only rarely.
The DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or stained in some way. A particularly sensitive method of staining DNA is to soak the gel in the dye ethidium bromide, which fluoresces under ultraviolet light when it is bound to DNA (see Figure 8–25B and D). Even more sensitive detection methods incorporate a radioisotope or chemical marker into the DNA molecules before electrophoresis, as we next describe.
Figure 8–25 DNA molecules can be separated by size using gel electrophoresis. (A) Schematic illustration comparing the results of cutting the same DNA molecule (in this case, the genome of a virus that infects wasps) with two different restriction nucleases, EcoRI (middle) and HindIII (right). The fragments are then separated by gel electrophoresis using a gel matrix of agarose. Because larger fragments migrate more slowly than smaller ones, the lowermost bands on the gel contain the smallest DNA fragments. The sizes of the fragments can be estimated by comparing them to a set of DNA fragments of known sizes (left). (B) Photograph of an actual agarose gel showing DNA “bands” that have been stained with ethidium bromide.
A polyacrylamide gel with small pores was used to separate short DNA molecules that differ by only a single nucleotide. Shown here are the results of a dideoxy sequencing reaction, explained later in this chapter. From left to right, the bands in the four lanes were produced by adding G, A, T, and C chain-terminating nucleotides (see Panel 8–1). The DNA molecules were labeled with 32P, and the image shown was produced by laying a piece of photographic film over the gel and allowing the 32P to expose the film, producing the dark bands observed when the film was developed.