A first step in the molecular analysis of a DNA segment or gene is to isolate it from the remainder of the DNA. A key moment in the development of molecular genetic methods was the discovery in the late 1960s of restriction enzymes (also called restriction endonucleases), which recognize and make double-stranded cuts in DNA at specific nucleotide sequences. These enzymes are produced naturally by bacteria and are used in defense against viruses. A bacterium protects its own DNA from a restriction enzyme by modifying the recognition sequence, usually by adding methyl groups to its DNA.

More than 800 different restriction enzymes that recognize and cut DNA at more than 100 different sequences have been isolated from bacteria. Many of these enzymes are commercially available; examples of some commonly used restriction enzymes are given in Table 14.1. The name of each restriction enzyme begins with an abbreviation that signifies its bacterial origin.

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The sequences recognized by restriction enzymes are usually from 4 to 8 bp long; most enzymes recognize a sequence of 4 or 6 bp. Most recognition sequences are palindromic—sequences that read the same (5′ to 3′) on the two complementary DNA strands.

Some restriction enzymes make staggered cuts in the DNA. For example, HindIII recognizes the following sequence:

HindIII cuts the sugar–phosphate backbone of each strand at the point indicated by the arrow, generating fragments with short, single-stranded overhanging ends:

Such ends are called cohesive ends, or sticky ends, because they are complementary to each other and can spontaneously pair to connect the fragments. Thus, DNA fragments with sticky ends can be “glued” together: any two fragments cleaved by the same enzyme will have complementary ends and will pair (Figure 14.1). When their cohesive ends have paired, the DNA fragments can be joined together permanently by DNA ligase, which seals nicks between the sugar–phosphate groups of the fragments.

Not all restriction enzymes produce staggered cuts and sticky ends (see Figure 14.1a). PvuII cuts in the middle of its recognition sequence, and the cuts on the two strands are directly opposite one another, producing blunt-ended fragments:

Fragments that have blunt ends are usually joined together in other ways.

The sequences recognized by a restriction enzyme are located randomly within the genome. Consequently, there is a relation between the length of the recognition sequence and the number of times it is present in a genome: there will be fewer longer recognition sequences than shorter recognition sequences because the probability of the occurrence of a particular sequence consisting of, say, six specific bases is less than the probability of the occurrence of a particular sequence of four specific bases. Therefore, restriction enzymes that recognize longer sequences will cut a given piece of DNA into fewer and longer fragments than will restriction enzymes that recognize shorter sequences.

Restriction enzymes are used whenever DNA fragments must be cut or joined. In a typical restriction reaction, a concentrated solution of purified DNA is placed in a small tube with a buffer solution and a small amount of restriction enzyme. Within a few hours, the enzyme cuts the appropriate restriction sites in all the DNA molecules, producing a mixture of DNA fragments.

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In recent years, geneticists have designed more complex restriction enzymes, termed engineered nucleases, that are capable of making double-stranded cuts to the DNA at any predetermined DNA sequence. Engineered nucleases consist of the part of a restriction enzyme that cleaves the DNA, coupled with another protein that recognizes and binds to a specific DNA sequence; the particular sequence to which the protein binds is determined by the protein’s amino acid sequence. By altering the amino acid sequence of the binding protein, geneticists can engineer a nuclease to bind to and cut any particular DNA sequence. Engineered nucleases include zinc finger nucleases (ZFNs), which use a DNA binding protein called a zinc finger, and transcription activator–like effector nucleases (TALENs), which use a protein that normally binds to sequences in promoters.

Another recently developed genome manipulation technique uses CRISPR RNAs, which are small RNAs found in bacteria and archaea (see Chapter 10). The CRISPR RNAs are coupled with nucleases that cut DNA. The CRISPR RNA binds to a specific DNA sequence, and its associated nuclease then cleaves the DNA at the recognized sequence. TRY PROBLEM 19