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The products of one 3-step cycle of PCR

Figure 9.23 Steps of a Single Cycle of PCR

The products of one 3-step cycle of PCR

Figure 9.23 Steps of a Single Cycle of PCR

two double-stranded DNA molecules for every original double-stranded target; after the next cycle, there will be four; after the next cycle there will be eight, and so on.

A critical factor in PCR is the heat-stable DNA polymerase of a thermophile, Thermus aquaticus. Taq polymerase, unlike the DNA polymerase of E. coli, is not destroyed at the high temperature used to denature the DNA in the first step of each amplification cycle. If a heat-stable polymerase were not used, fresh polymerase would need to be added for every cycle of the reaction. Thus, the discovery and characterization of T. aquaticus through basic research was instrumental in developing this widely used and commercially valuable method. ■ thermophile, p. 87

Generating a Discrete-Sized Fragment

While the preceding description explains how PCR amplifies the target DNA exponentially, it does not clarify how a discrete-length fragment becomes the ultimate product. The generation of fragments of a particular size is important, because it enables a researcher to use PCR to readily detect the presence of target DNA in a sample. After PCR, the amplified target can be viewed as a single band on the ethidium bromide-stained gel.

To understand how discrete-sized fragments of target DNA are generated, you must consider the exact sites to which the primers anneal and visualize at least three cycles of replication (figure 9.24). In the first cycle, two new fragments are generated. Note, however, that these fragments are shorter than the original full-length template molecules but longer than the target DNA. Their 5' end is primer DNA. These mid-length products will be generated whenever the original full-length molecule is used as a template, which is one time each replication cycle.

In the next cycle, the full-length molecules will again be used as templates, repeating the process just described. More importantly, the mid-length fragments created during the first cycle will be used as templates for DNA synthesis. As before, the primers will anneal to these fragments and then nucleotides will be added to the 3' end. Elongation, however, will stop at the 5' end of the template molecule, because DNA synthesis requires a template. Recall that the 5' end of the template is primer DNA. Thus, whenever a mid-length fragment is used as a template, a short fragment is generated. The 5' and 3' ends of this fragment are determined by the sites to which the primers initially annealed.

In the third round of replication, the full-length and the mid-length fragments again will be used as templates, repeating the processes just described. The short fragments generated in the preceding round will also be used as templates, however, generating short double-stranded molecules. Continuing to follow the events in further rounds of replication will reveal that it is this fragment that is exponentially amplified. Ultimately, enough of these short fragments are generated to be detectable using gel electrophoresis followed by ethidium bromide staining.

Nester-Anderson-Roberts: I I. Life and Death of I 9. Biotechnology and I I © The McGraw-Hill

Microbiology, A Human Microorganisms Recombinant DNA Companies, 2003

Perspective, Fourth Edition

Cycle 1

Cycle 2

Cycle 3

Target DNA _I_

Template

Mid-length fragment

Primer

Primerx , Mid-length fragment

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