DNA that exists in the nuclei of your cells is present in two copies per cell, except for DNA in the X and Y chromosomes in males, which are each present in only one copy per cell in males. In the laboratory, it is very difficult to manipulate or visualize a sample containing just one or two copies of a DNA molecule. Instead, researchers typically work with many identical copies of the DNA molecule they are interested in. A common method for making copies of a piece of DNA is the polymerase chain reaction (PCR), which allows a targeted region of a DNA molecule to be replicated (or amplified) into as many copies as desired. PCR is both selective and highly sensitive, so it is used to amplify and detect small quantities of nucleic acids, such as HIV in blood-bank supplies, or to study DNA samples as minuscule as those left by a smoker’s lips on a cigarette butt dropped at the scene of a crime. The starting sample can be as small as a single molecule of DNA.

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The principles of PCR are illustrated in Fig. 12.13. Because the PCR reaction is essentially a DNA synthesis reaction, it requires the same basic components used by the cell to replicate its DNA. In this case, the procedure takes place in a small plastic tube containing a solution that includes four essential components:

The primer sequences are oligonucleotides (oligos is the Greek word for “few”) produced by chemical synthesis and are typically 20–30 nucleotides long. Their base sequences are chosen to be complementary to the ends of the region of DNA to be amplified. In other words, the primers flank the specific region of DNA to be amplified. The 3′ end of each primer must be oriented toward the region to be amplified so that, when DNA polymerase extends the primer, it creates a new DNA strand complementary to the targeted region. Because the 3′ ends of the primers both point toward the targeted region, one of the primers pairs with one strand of the template DNA and the other pairs with the other strand of the template DNA.

PCR creates new DNA fragments in a cycle of three steps, as shown in Fig. 12.13a. The first step, denaturation, involves heating the solution in a plastic tube to a temperature just short of boiling so that the individual DNA strands of the template separate (or “denature”) as a result of the breaking of hydrogen bonds between the complementary bases. The second step, annealing, begins as the solution is cooled. Because of the great excess of primer molecules, the two primers bind (or “anneal”) to their complementary sequence on the DNA (rather than two strands of the template duplex coming back together). In the final step, extension, the solution is heated to the optimal temperature for DNA polymerase and the polymerase elongates (or “extends”) each primer with the deoxynucleoside triphosphates.

After sufficient time to allow new DNA synthesis, the solution is heated again, and the cycle of denaturation, annealing, and extension is repeated over and over, as indicated in Fig. 12.13b, usually for 25–35 cycles. In each cycle, the number of copies of the targeted fragment is doubled. The first round of PCR amplifies the targeted region into 2 copies, the next into 4, the next into 8, then 16, 32, 64, 128, 256, 512, 1024, and so forth. By the third round of amplification, the process begins to produce molecules that are only as long as the region of the template duplex flanked by the sequences complementary to the primers. After several more rounds of replication the majority of molecules are of this type. The doubling in the number of amplified fragments in each cycle justifies the term “chain reaction.”

Although PCR is elegant in its simplicity, the DNA polymerase enzymes from many species (including humans) irreversibly lose both structure and function at the high temperature required to separate the DNA strands. At each cycle, you would have to open the tube and add fresh DNA polymerase. This is possible, and in fact it was how PCR was done when the technique was first developed, but the procedure is time consuming and tedious. To solve this problem, we now use DNA polymerase enzymes that are heat-stable, such as those from the bacterial species Thermus aquaticus, which lives at the near–boiling point of water in natural hot springs, including those at Yellowstone National Park. This polymerase, called Taq polymerase, remains active at high temperatures. Once the reaction mixtures are set up, the entire procedure is carried out in a fully automated machine. The time of each cycle, temperatures, number of cycles, and other variables can all be programmed. The fact that DNA polymerase from a bacterium that lives in hot springs can be used to amplify DNA from any organism reminds us again of the conserved function and evolution early in the history of life of this enzyme.