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| Modern Genetic Analysis Chapter 10. Recombinant DNA Technology Using Cloned DNA Figure 10-26. The polymerase chain reaction. (a) Double-stranded DNA containing the target sequence. (b) Two chosen or created primers have sequences complement-ing primer-binding sites at the 3 ends of the target gene on the two strands. The strands are separated by heating, allowing the two primers to anneal to the primer-binding sites. Together, the primers thus flank the targeted sequence. (c) Taq polymerase then synthesizes the first set of complementary strands in the reaction. These first two strands are of varying length, because they do not have a common stop signal. They extend beyond the ends of the target sequence as delineated by the primer-binding sites. (d) The two duplexes are heated again, exposing four binding sites. (For simplicity, only the two new strands are shown.) The two primers again bind to their respective strands at the 3 ends of the target region. (e) Taq polymerase again synthesizes two complementary strands. Although the template strands at this stage are variable in length, the two strands just synthesized from them are precisely the length of the target sequence desired. This is because each new strand begins at the primer-binding site, at one end of the target sequence, and proceeds until it runs out of template, at the other end of the sequence. (f) Each new strand now begins with one primer sequence and ends with the primer-binding sequence for the other primer. Subsequent to strand separation, the primers again anneal and the strands are extended to the length of the target sequence. (The variable-length strands from part c are also producing target-length strands.) (g) The process can be repeated indefinitely, each time creating two double-stranded DNA molecules identical with the target sequence. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright © 1992 by Scientific American Books.)

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