Fig. 8. The polymerase chain reaction (PCR). Amplification of a small amount of DNA from any of several different tissue sources by PCR assay has proved to be an invaluable aid to modern molecular biology and genetics. The steps in the PCR process are shown here. The reactions use several different temperatures to allow first denaturation, melting, of DNA strands from their normal double-stranded state into single strands. The second step is to anneal synthetic primers that bind to the melted DNA. The third step is to elongate and copy the template strands into new DNA in an elongation step. We cycle among the three steps at three different temperatures. Each temperature favors a different property: denaturation at 94°C, annealing at about 55°C, and elongation at 72°C. Usually 25 to 40 cycles are sufficient to synthesize enough DNA for easy visualization of the product by staining with ethidium bromide. In cycles beyond the third one, the PCR accumulates the desired product in exponential fashion. The first three cycles accumulate some newly synthesized DNA strands that extend beyond the end of one or the other primer, but these are of little consequence at the end of the amplification. |