Unlike a protein, a gene does not exist as a discrete entity in cells, but rather as a small region of a much longer DNA molecule. Although the DNA molecules in a cell can be randomly broken into small pieces by mechanical force, a fragment containing a single gene in a mammalian genome would still be only one among a hundred thousand or more DNA fragments, indistinguishable in their average size. How could such a gene be separated from all the others? Because all DNA molecules consist of an approximately equal mixture of the same four nucleotides, they cannot be readily separated, as proteins can, on the basis of their different charges and biochemical properties. The solution to this problem began to emerge with the discovery of restriction nucleases. These enzymes, which are purified from bacteria, cut the DNA double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long, double-stranded DNA molecule into fragments of strictly defined sizes.
Like many of the tools of recombinant DNA technology, restriction nucleases were discovered by researchers trying to understand an intriguing biological phenomenon. It had been observed that certain bacteria always degraded “foreign” DNA that was introduced into them experimentally. A search for the mechanism responsible revealed a then unanticipated class of bacterial nucleases that cleave DNA at specific nucleotide sequences. The bacterium’s own DNA is protected from cleavage by methylation of these same sequences, thereby protecting a bacterium’s own genome from being overrun by foreign DNA. Because these enzymes restrict the transfer of DNA into bacteria, they were called restriction nucleases. The pursuit of this seemingly arcane biological puzzle set off the development of technologies that have forever changed the way cell and molecular biologists study living things.
Different bacterial species produce different restriction nucleases, each cutting at a different, specific nucleotide sequence (Figure 8–24). Because these target sequences are short—generally four to eight nucleotide pairs—many sites of cleavage will occur, purely by chance, in any long DNA molecule. The reason restriction nucleases are so useful in the laboratory is that each enzyme will always cut a particular DNA molecule at the same sites. Thus for a given sample of DNA (which contains many identical molecules), a particular restriction nuclease will reliably generate the same set of DNA fragments.
The size of the resulting fragments depends on the length of the target sequences of the restriction nucleases. As shown in Figure 8–24, the enzyme HaeIII cuts at a sequence of four nucleotide pairs; a sequence this long would be expected to occur purely by chance approximately once every 256 nucleotide pairs (1 in 44). In comparison, a restriction nuclease with a target sequence that is eight nucleotides long would be expected to cleave DNA on average once every 65,536 nucleotide pairs (1 in 48). This difference in sequence selectivity makes it possible to cleave a long DNA molecule into the fragment sizes that are most suitable for a given application.
Gel Electrophoresis Separates DNA Molecules of Different Sizes
The same types of gel-electrophoresis methods that have proved so useful in the analysis of proteins (see Figure 8–13) can be applied to DNA molecules. The procedure is actually simpler than for proteins: because each nucleotide in a nucleic acid molecule already carries a single negative charge (on the phosphate group), there is no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode. Larger DNA fragments will migrate more slowly because their progress is impeded to a greater extent by the gel matrix. Over several hours, the DNA fragments become spread out across the gel according to size, forming a ladder of discrete bands, each composed of a collection of DNA molecules of identical length (Figure 8–25A and B). To separate DNA molecules longer than 500 nucleotide pairs, the gel is made of a diluted solution of agarose (a polysaccharide isolated from seaweed). For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels allow the separation of molecules that differ in length by as little as a single nucleotide (see Figure 8–25C).
Figure 8–24 Restriction nucleases cleave DNA at specific nucleotide sequences.