19.1.8—PCR to clone and amplify DNA

Polymerase chain reaction (PCR) is an in-vitro method for making many copies of a specific DNA target from a very small starting sample. Primers define which region is copied, while a thermostable DNA polymerase builds the new strands during repeated temperature cycles.

The reaction needs target DNA, forward and reverse primers, free nucleotides, a DNA polymerase, and a buffer. Primers are complementary to the ends of the target region, so they provide specificity and tell the polymerase where to begin. Taq polymerase is useful because it remains active after the high-temperature denaturation stage.

1. Denaturation: heat the double-stranded template so hydrogen bonds break and the strands separate (about 95°C in the SME method).
2. Annealing: lower the temperature so the forward and reverse primers bind to complementary sequences at the target ends (about 50–60°C).
3. Extension: raise the temperature to the polymerase optimum so complementary strands are built from free nucleotides (about 72°C for Taq).
4. Repeat the cycle: each cycle doubles the target DNA, producing exponential amplification of the region between the primers.

PCR amplifies the sequence between the primers, not every DNA molecule in the sample. Specificity therefore depends on primer binding and reaction conditions; a large signal is not automatically proof that the intended biological interpretation is correct. Contamination-control procedures are not specified on the matched SME page, so no extra protocol is invented here. Gel electrophoresis, microarrays and bioinformatics are separate cards.