Polymerase Chain Reaction

Mark V. Bloom, Ph.D.
DNA Learning Center,
Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York 11724

Louis Pasteur once remarked that "chance favors the prepared mind," and certainly the history of scientific progress supports his contention. The annals of science provide numerous examples of serendipitous discovery. Some are mythic, such as Newton's discovery of gravity following his encounter with an apple; while others are more rooted in fact - like Fleming's discovery of penicillin on a contaminated petri dish. Scientists today continue to take unexpected turns on their paths to discovery. One such recent detour occurred in 1983 on U.S. Route 101 in northern California. Kary Mullis (Fig. 1), a scientist working for the Cetus Corporation, was driving along the mountain road with a friend one moonlit night. His mind constantly shifted from the road to a problem of nucleic acid biochemistry. He was struggling to devise a simple method for determining the identity of a specific nucleotide along a stretch of DNA. It seemed that just as he solved one technical problem, another one took its place. Suddenly, a flash of insight caused him to pull the car off the road and stop. He awakened his friend dozing in the passenger seat and excitedly explained to her that he had hit upon a solution - not to his original problem, but to one of even greater significance. Kary Mullis had just conceived of a simple method for producing virtually unlimited copies of a specific DNA sequence in a test tube - the polymerase chain reaction (PCR). Figure 1 Kary Mullis The polymerase chain reaction was introduced to the scientific community at a conference in October 1985. Scientists, quick to embrace the new technique, were surprised (with the wisdom that accompanies hindsight) that no one had thought of it earlier. Cetus rewarded Kary Mullis with a $10,000 bonus for his invention, and later, during a corporate reorganization, sold the patent for the PCR process to the pharmaceutical company Hoffmann-La Roche for $300 million! The popularity of PCR continues unabated. As of the end of 1993, PCR has been referenced in well over 7,000 scientific publications.

DNA Hybridization The chemistry of PCR, as with much of molecular biology, depends on the complementarity of the DNA bases. When a molecule of DNA is sufficiently heated, the hydrogen bonds holding together the double helix are disrupted and the molecule unzips or "denatures" into single strands. If the DNA solution is allowed to cool, then complementary base pairs can reform (renature) and the original double helix is restored. Experiments performed during the 1960s demonstrated that many DNA sequences were not unique within the genome. Purified DNA solutions were denatured by heat and then allowed to cool. Using a spectrophotometer it was possible to monitor the rate at which the DNA renatured. Data from these studies revealed that genomes are composed of different classes of DNA sequences that can be distinguished by their repetitive frequency. For example, some amphibian cells contain more DNA than human cells, owing to a large excess of repetitive DNA relative to their human counterparts. While useful for studying the broad outline of genome organization, this approach could not be used to investigate the structure of individual genes. This ability came about during the 1970s following the introduction of DNA restriction analysis and nucleic acid hybridization techniques. Hybridization allows a specific DNA sequence to be analyzed against the complex background of a eukaryotic genome. It is estimated that the human genome contains between 100,000 and 200,000 genes. To focus on an individual gene, DNA from the target organism is isolated, fragmented with restriction enzymes, and separated by gel electrophoresis. The DNA fragments are denatured to render them single stranded and exposed to a solution containing a radioactive DNA "probe." The probe consists of single-stranded nucleic acid (either DNA or RNA) with a sequence chosen to base pair with the gene of interest. Under appropriate conditions of temperature, salt, and pH, called "stringency," the probe will bind to its corresponding sequence in the target DNA and nowhere else. The presence of a radioactive signal (often by exposure to X-ray film) indicates positions of probe binding. Go to the next page...