Techniques Used in the Polymerase Chain Reaction PCR

The polymerase chain reaction (PCR) starts with a double-stranded DNA molecule, from which millions of identical copies of a select region can be produced. The process involves a sequential series of DNA synthesis reactions and the following key ingredients:

■ Double-stranded DNA containing the region to be amplified, the target DNA.

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■ A heat-stable DNA polymerase, Taq polymerase, which is from Thermus aquaticus.

■ Each of the four nucleotides (dATP, dGTP, dCTP, dTTP).

■ Short single-stranded segments of DNA, generally about 20 nucleotides in length, to serve as primers. The selection of these primers will be discussed shortly.

The Three-Step Amplification Cycle

PCR requires a repeating cycle consisting of three steps (figure 9.23). In the first step, the double-stranded DNA is denatured by heating the sample to approximately 95°C. In the second step, the temperature is lowered to approximately 50°C; within seconds, the primers anneal to their complementary sequences on the denatured target DNA. In the third step, DNA synthesis occurs when the temperature is raised to the optimal temperature for Taq DNA polymerase, approximately 70°C. The DNA polymerase adds nucleotides to the 3' end of the DNA primer using the opposing strand as a template. The net result is the synthesis of two new strands of DNA, each of which is complementary to the other. In other words, the three-step cycle results in the duplication of the original target DNA. Since each of the newly synthesized strands can then serve as a template strand for the next cycle, the DNA is amplified exponentially. After a single cycle of the three-step reaction, there will be

^eg ion of DNA to be ami plifie^

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