Figure 7-38. The polymerase chain reaction.

Figure 7-38The polymerase chain reaction

The starting material is a double-stranded DNA. Large numbers of primers are added, each with the sequence found in one strand at the end of the region to be amplified. The thermostable Taq polymerase and dNTPs are also added. In the first cycle, heating to 95 °C melts the double-stranded DNA and subsequent cooling to 60 °C then allows the excess primers to hybridize (anneal) to their complementary sequences in the target DNA. The Taq polymerase then extends each primer from its 3′ end by polymerization of dNTPs, generating newly synthesized strands (wavy lines) that extend in the 3′ direction to the 5′ end of the template restriction fragment. In the second cycle, the original and newly made DNA strands are separated at 95 °C and primers annealed to their complementary sequences at 60 °C. (For simplicity, subsequent events involving only newly made strands are shown; these soon greatly outnumber the original strands.) Each annealed primer again is extended by Taq polymerase to the end of the other primer sequence at the 5′ end of the template strand. Thus the strands (amplimers) synthesized in this cycle exactly equal the length of region to be amplified. In the third cycle, two double-stranded DNA molecules are generated equal to the sequence of the region to be amplified. These two are doubled in the fourth cycle and are doubled again with each successive cycle. [Adapted from J. D. Watson et al., 1992, Recombinant DNA, 2d ed., Scientific American Books.]

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From: Section 7.7, Polymerase Chain Reaction: An Alternative to Cloning

Cover of Molecular Cell Biology
Molecular Cell Biology. 4th edition.
Lodish H, Berk A, Zipursky SL, et al.
New York: W. H. Freeman; 2000.
Copyright © 2000, W. H. Freeman and Company.

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