circular, is used because this isolation is usually accomplished by making many identical copies of only the DNA of interest. We note that elsewhere in the book, cloning, particularly when used in the context of developmental biology, can also refer to the generation of many genetically identical cells starting from a single cell or even to the generation of genetically identical organisms (see, for example, Figure 7–2). In all cases, cloning refers to the act of making many identical copies, and in this section, we use the term to refer to methods designed to generate many identical copies of a defined segment of nucleic acid.
DNA cloning can be accomplished in several ways. One of the simplest involves inserting a particular fragment of DNA into the purified DNA genome of a self-replicating genetic element—usually a plasmid. The plasmid vectors most widely used for gene cloning are small, circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells. They generally account for only a minor fraction of the total host bacterial cell DNA, but owing to their small size, they can easily be separated from the much larger chromosomal DNA molecules, which precipitate as a pellet upon centrifugation. For use as cloning vectors, the purified plasmid DNA circles are first cut with a restriction nuclease to create linear DNA molecules. The DNA to be cloned is added to the cut plasmid and then covalently joined using the enzyme DNA ligase (Figure 8–27 and Figure 8–28). As discussed in Chapter 5, this enzyme is used by the cell to stitch together the Okazaki fragments produced during DNA replication. The recombinant DNA circle is introduced back into bacterial cells that have been made transiently permeable to DNA. As the cells grow and divide, doubling in number every 30 minutes, the recombinant plasmids also replicate to produce an
Figure 8–27 The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA ligase. The plasmid is cut open with a restriction nuclease (in this case, one that produces staggered ends) and is mixed with the DNA fragment to be cloned (which has been prepared with the same restriction nuclease). DNA ligase and ATP are added. The staggered ends base-pair, and DNA ligase seals the nicks in the DNA backbone, producing a complete recombinant DNA molecule. In the accompanying micrographs, the inserted DNA is colored red. (Micrographs courtesy of Huntington Potter and David Dressler.)
Figure 8–28 DNA ligase can join together any two DNA fragments in vitro to produce recombinant DNA  molecules. ATP provides the energy  necessary to reseal the sugar-phosphate  backbone of DNA (see Figure 5–12).
DNA ligase can readily join two  DNA fragments produced by the same restriction nuclease, in this case EcoRI. Note that the staggered ends produced by this enzyme enable the ends of the two fragments to base-pair correctly with each other, greatly facilitating their rejoining.
DNA ligase can also be used to join DNA fragments produced by different restriction nucleases—for example, EcoRI and HaeIII. In this case, before the fragments undergo ligation, DNA polymerase plus a mixture of deoxyribonucleoside triphosphates (dNTPs) are used to fill in the staggered cut produced by EcoRI. Each DNA fragment shown in the figure is oriented so that its 5ʹ ends are at the left end of the upper strand and the right end of the lower strand, as indicated.
cell culture produces hundreds of millions of new bacteria enormous number of copies of DNA circles containing the foreign DNA (Figure 8–29). Once the cells are lysed and the plasmid DNA isolated, the cloned DNA fragment can be readily recovered by cutting it out of the plasmid DNA with the same restriction nuclease that was used to insert it, and then separating it from the plasmid DNA by gel electrophoresis. Together, these steps allow the amplification and purification of any segment of DNA from the genome of any organism.