In step 1, a starting solution of DNA is heated to between 90° and 100°C to break the hydrogen bonds between the two nucleotide strands and thus produce the necessary single-stranded templates. The reaction mixture is held at this temperature for only a minute or two. In step 2, the DNA solution is cooled quickly to between 30° and 65°C and held at this temperature for a minute or less. During this short interval, the DNA strands will not have a chance to reanneal, but the primers will be able to attach to their complementary sequences on the template strands. In step 3, the solution is heated to between 60° and 70°C, the temperature at which DNA polymerase can synthesize new DNA strands by adding nucleotides to the primers. Within a few minutes, two new double-stranded DNA molecules are produced for each original molecule of target DNA.

The whole cycle is then repeated. With each cycle, the amount of target DNA doubles; so the target DNA increases geometrically. One molecule of DNA increases to more than 1000 molecules in 10 PCR cycles, to more than 1 million molecules in 20 cycles, and to more than 1 billion molecules in 30 cycles (Table 18.5). Each cycle is completed within a few minutes; so a large amplification of DNA can be achieved within a few hours.

Two key innovations facilitated the use of PCR in the laboratory. The first was the discovery of a DNA poly-merase that is stable at the high temperatures used in step 1 of PCR. The DNA polymerase from E. coli that was originally used in PCR denatures at 90°C. For this reason, fresh enzyme had to be added to the reaction mixture during each cycle, slowing the process considerably. This obstacle was overcome when DNA polymerase was isolated from the bacterium Thermus aquaticus, which lives in the boiling springs of Yellowstone National Park (see Figure 18.18). This enzyme, dubbed Taq polymerase, is remarkably stable at high temperatures and is not denatured during the strand-separation step of PCR; so it can be added to the reaction mixture at the beginning of the PCR process and will continue to function through many cycles.

The second key innovation was the development of automated thermal cyclers—machines that bring about the rapid temperature changes necessary for the different steps of PCR. Originally, tubes containing reaction mixtures were moved by hand among water baths set at the different temperatures required for the three steps of each cycle. In automated thermal cyclers, the reaction tubes are placed in a metal block that changes temperature rapidly according to a computer program.

The polymerase chain reaction is now often used in place of gene cloning, but it does have several limitations. First, the use of PCR requires prior knowledge of at least part of the sequence of the target DNA to allow construction of the primers. Therefore PCR cannot be used to amplify a gene that has not been at least partly sequenced. Second, the capacity of PCR to amplify extremely small amounts of DNA makes contamination a significant problem. Minute amounts of DNA from the skin of laboratory workers and even in small particles in the air can enter a reaction tube and be amplified along with the target DNA. Careful laboratory technique and the use of controls are necessary to circumvent this problem.

A third limitation of PCR is accuracy. Unlike other DNA polymerases, Taq polymerase does not have the capacity to proofread (see p. 000 in Chapter 12) and, under standard PCR conditions, it incorporates an incorrect nucleotide about once every 20,000 bp. DNA polymerases with proofreading capacity usually incorporate an incorrect nucleotide only about once every billion base pairs. For many applications, the error rate produced by PCR is not a problem, because only a few DNA molecules of the billions produced will contain an error. However, for other applications such as the cloning of PCR products, the relatively high error rate of PCR can pose significant problems. New heat-stable DNA polymerases with proofreading capacity have been isolated, giving more accurate PCR results.

A fourth limitation of PCR is that the size of the fragments that can be amplified by standard Taq polymerase is usually less than 2000 bp. By using a combination of Taq polymerase and a DNA polymerase with proofreading capacity and by modifying the reaction conditions, investigators have been successful in extending PCR amplification to larger fragments. In spite of its limitations, PCR is used routinely in a wide array of molecular applications.

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