28.8 • The Polymerase Chain Reaction
It often happens that only a tiny amount of DNA can be obtained directly, as might occur at a crime scene, so methods for obtaining larger amounts are sometimes needed to carry out sequencing and characterization. The invention of the polymerase chain reaction (PCR) by Kary Mullis in 1986 has been described as being to genes what Gutenberg’s invention of the printing press was to the written word. Just as the printing press produces multiple copies of a book, PCR produces multiple copies of a given DNA sequence. Starting from less than 1 picogram (pg) of DNA with a chain length of 10,000 nucleotides (1 pg = 10–12 g; about 105 molecules), PCR makes it possible to obtain several micrograms (1 μg = 10–6 g; about 1011 molecules) in just a few hours.
The key to the polymerase chain reaction is Taq DNA polymerase, a heat-stable enzyme isolated from the thermophilic bacterium Thermus aquaticus found in a hot spring in Yellowstone National Park. Taq polymerase is able to take a single strand of DNA having a short, primer segment of complementary chain at one end and then finish constructing the entire complementary strand. The overall process takes three steps, as shown in Figure 28.10. More recently, improved heat-stable DNA polymerases have become available, including Vent polymerase and Pfu polymerase, both isolated from bacteria growing near geothermal vents in the ocean floor. The error rate of both enzymes is substantially less than that of Taq.
The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion, the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands.
The temperature is lowered to between 37 and 50 °C, allowing the primers, because of their relatively high concentration, to anneal by hydrogen-bonding to their complementary sequence at the end of each target strand.
The temperature is then raised to 72 °C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands. When replication of each strand is complete, two copies of the original DNA now exist. Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, repeating a third time yields eight copies, and so on, in an exponential series.
PCR has been automated, and 30 or so cycles can be carried out in an hour, resulting in a theoretical amplification factor of 230 (∼109). In practice, however, the efficiency of each cycle is less than 100%, and an experimental amplification of about 106 to 108 is routinely achieved for 30 cycles.