Recombinant DNA techniques, developed in the early 1970s, have taken biology from an exclusively analytical science to a synthetic one. New combinations of unrelated genes can be constructed in the laboratory by applying recombinant DNA techniques. These novel combinations can be cloned—amplified many times—by introducing them into suitable cells, where they are replicated by the DNA-synthesizing machinery of the host. The inserted genes are often transcribed and translated in their new setting, producing proteins that would not ordinarily be found in the host cell. Recall also that recombinant DNA techniques allow the researcher to remove (knock-out) or add (knock-in) specific genes from or to a genome.

Restriction enzymes are perhaps the tools that made the development of recombinant DNA technology possible. Restriction enzymes, also called restriction endonucleases, recognize specific base sequences in double-helical DNA and cleave, at specific places, both strands of that duplex. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned.

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Restriction enzymes are found in a wide variety of bacteria. Their biological role is to cleave and thereby destroy foreign DNA molecules, such as the DNA of viruses that attack bacteria (bacteriophages). The cell’s own DNA is not degraded, because the sites recognized by its own restriction enzymes are methylated, which prevents DNA cleavage. Many restriction enzymes recognize specific sequences of four to eight base pairs, called cleavage sites, and hydrolyze a phosphodiester linkage at a specific site in each strand in this region. A striking characteristic of these cleavage sites is that they almost always possess twofold rotational symmetry. In other words, the recognized sequence is palindromic, or an inverted repeat, and the cleavage sites are symmetrically positioned. For example, the sequence recognized by a restriction enzyme from Streptomyces achromogenes is

In each strand, the enzyme cleaves the C–G phosphodiester linkage on the 3′ side of the symmetry axis.

Hundreds of restriction enzymes have been purified and characterized. Their names consist of a three-letter abbreviation for the host organism (e.g., Eco for Escherichia coli, Hin for Haemophilus influenzae, Hae for Haemophilus aegyptius), followed by a strain designation (if needed) and a roman numeral (if more than one restriction enzyme from the same strain has been identified). The specificities of several of these enzymes are shown in Figure 41.2. Restriction enzymes are used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire parent molecule.

Small differences between related DNA molecules can be readily detected because their restriction fragments can be separated and displayed by gel electrophoresis. In Chapter 5, we considered the use of gel electrophoresis to separate protein molecules. Gel electrophoresis of nucleic acids is similar in principle to gel electrophoresis of proteins. When working with nucleic acids, however, the sample is not denatured and the gel is often made of agarose instead of polyacrylamide. Because the phosphodiester backbone of DNA is highly negatively charged, this technique is also suitable for the separation of nucleic acid fragments. For most gels, the shorter the DNA fragment, the farther the migration. Polyacrylamide gels are used to separate, by size, fragments containing as many as 1000 base pairs, whereas more porous agarose gels are used to resolve mixtures of larger fragments (as large as 20 kb). An important feature of these gels is their high resolving power. In certain kinds of gels, fragments differing in length by just one nucleotide of several hundred can be distinguished. Bands or spots of DNA in gels can be visualized by staining with a dye such as ethidium bromide, which fluoresces an intense orange under irradiation by ultraviolet light when bound to a double-helical DNA molecule (Figure 41.3). A band containing only 50 ng of DNA can be readily seen.

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A restriction fragment containing a specific base sequence can be identified by hybridizing it with a labeled complementary DNA strand, such as our probe for the estrogen receptor (Figure 41.4). A mixture of restriction fragments is separated by electrophoresis through an agarose gel, denatured to form single-stranded DNA, and transferred to a nitrocellulose sheet. The positions of the DNA fragments in the gel are preserved on the nitrocellulose sheet, where they are exposed to a ³²P-labeled single-stranded DNA probe. A sheet of x-ray film is then placed over the blot. The radioactivity of the probe will expose (darken) the x-ray film. This process, called autoradiography, reveals the position of the restriction-fragment–probe duplex. A particular fragment amid a million others can be readily identified in this way. This powerful technique is named Southern blotting, for its inventor Edwin Southern.

Similarly, RNA molecules can be separated by gel electrophoresis, and ­specific sequences can be identified by hybridization subsequent to their transfer to nitrocellulose. This analogous technique for the analysis of RNA has been whimsically termed northern blotting. A further play on words accounts for the term western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody (Chapter 5). Southern, northern, and western blots are also known respectively as DNA, RNA, and protein blots.

Let us examine how novel DNA molecules can be constructed in the laboratory as preparation for isolating DNA encoding the estrogen receptor. Our immediate goal is to insert DNA encoding the receptor into a piece of DNA, called a vector, that is readily taken up and replicated by bacteria. The bacteria containing the foreign DNA can be isolated, or cloned. The cloned bacteria can then produce large amounts of receptor proteins with which we can perform experiments.

How do we construct a recombinant DNA molecule? A DNA fragment of interest is covalently joined to a DNA vector. The essential feature of a vector is that it can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in bacteria) and bacteriophage lambda (λ phage), a virus, are commonly used vectors for cloning in E. coli. The vector can be prepared for accepting a new DNA fragment by cleavage at a single specific site with a restriction enzyme. The staggered cuts made by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are known as cohesive or sticky ends. Any DNA fragment can be inserted into this plasmid if the fragment has the same cohesive ends. Such a fragment can be prepared from a larger piece of DNA by using the same restriction enzyme as was used to open the plasmid DNA (Figure 41.5).

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The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid can be annealed and then joined by DNA ligase.