4 - The Polymerase Chain Reaction

Abstract

A typical polymerase-chain-reaction (PCR) program consists of a denaturing step, an annealing step, and an extension step. The requirements for a PCR are a thermostable deoxyribonucleic acid (DNA) polymerase, a small amount of initial DNA (template), and two suitable oligonucleotide primers. The reaction begins after adding buffers and nucleotides to this and placing the tube in a PCR machine. Denaturing, whereby the two strands of the DNA template are separated from one another, is carried out at 94°C. The temperature is subsequently lowered to 55°C so that a hybridization of the oligonucleotide primer, which is available in massive excess, is carried out on the single-stranded DNA template. After that, the temperature is increased to 72°C, the optimal temperature for Taq polymerase. The primer is thereby elongated until a double-stranded DNA is formed, which is exactly equivalent to the original DNA template. Because complementation occurs along both strands of the DNA template, the number of template DNA is doubled in the course of one cycle. If this cycle is repeated, the experimenter obtains a fourfold quantity. By means of nested PCR, extremely small quantities of template can be demonstrated. This method allows the amplification of almost every complementary DNA (cDNA) from any desirable tissue. The uses of PCR appear to be endless—at least there does not seem to be any end to this ingenuity at present.