After his epiphanous conception of the polymerase chain reaction while driving on a California highway, Kary B. Mullis, along with his colleagues at Cetus Corporation, successfully developed this powerful new technique for the rapid in vitro manufacture of massive amounts of DNA from small samples, which led to valuable applications in medicine, genetics, criminology, archaeology, and other fields.
Ever since the British biophysicist Francis Crick
During the 1980’s, problems about the safety of recombinant DNA research were solved, and an intensely competitive relationship among the new biotechnology companies soon evolved. Cetus Corporation, which had been founded in 1971, originally concentrated on synthesizing short chains of DNA for scientists to use in their research on genetic cloning, but in 1976, when Cetus hired David Gelfand, the focus shifted to recombinant DNA research. When the company went public in 1981, the influx of capital facilitated Cetus’s new direction.
Kary B. Mullis, who had received his Ph.D. in biochemistry from the University of California, Berkeley, in 1972, joined Cetus in 1979, after postdoctoral work in pharmaceutical chemistry at the University of California, San Francisco. Thomas J. White, who had known Mullis at Berkeley, convinced David Gelfand to hire Mullis as a DNA chemist, and Mullis, who had become interested in DNA synthesizing machines, accepted the position. In his early days at Cetus, Mullis acquired a reputation for wild ideas that his colleagues paid little attention to. Undaunted, he became intrigued by iterative processes, and he began exploring exponential amplification on computers both at work and at home.
According to Mullis, the “eureka moment” in his discovery of the polymerase chain reaction (PCR) occurred during a drive in the spring of 1983 through Mendocino County, California. While his girlfriend, Jennifer Barnett, was asleep beside him, he began thinking about DNA, enzymes, and other chemicals he had been working on. The DNA molecule is a polynucleotide that is, it is made up of a large number of nucleotides, which are compounds of a sugar (deoxyribose), a base (a purine or a pyrimidine), and a phosphate group. Because the DNA molecule is so large, chemists often work with small sections of it consisting of two or more nucleotides, called oligonucleotides. Mullis had made oligonucleotides in his laboratory at Cetus. Since a DNA molecule typically has three billion nucleotides, chemists often encounter problems in finding a specific DNA sequence and then making it available for study or use. Mullis got the idea that if he could construct a short section of artificial DNA and then mesh it with a particular segment of natural DNA, then he could start a process in which that segment would reproduce itself over and over again. DNA had a natural replicative ability, and Mullis hoped to capitalize on this power by amplifying a target sequence many fold in a short time.
Mullis was so excited by this idea that he pulled off the road, noting exactly where he parked. He made some preliminary calculations, confirming that further cycles of DNA replication would yield billions of copies, each copy having the same size. His simple technique was selective (he could replicate any DNA sequence he chose) and superabundant (the chain reaction led to an exponential growth of product). He felt then that his idea was significant enough to win him a Nobel Prize, if he could get it to work in the laboratory.
After his return to Cetus, Mullis searched the scientific literature and found that no one had anticipated his technique for the amplification of DNA sequences. To his surprise, however, none of his colleagues at Cetus took interest in his idea. However, Ron Cook,
In June, 1984, Mullis returned to the fifty-eight-base-pair region of the gene for human beta-globin that contained the sickle-cell anemia mutation, and he achieved some amplification of this DNA segment. Despite this progress, he needed to find the best materials, temperatures, and procedures for maximizing amplification. He discovered that the specificity of amplification depended on the design of primers, small pieces of synthetic DNA that can bind to each end of a targeted natural DNA sequence. The multiplication of this sequence depended on DNA polymerase, a naturally occurring enzyme that catalyzes the formation of DNA. Mullis found that Taq polymerase, which had been discovered in the hot springs of Yellowstone National Park (hence its full name, Thermophilus aquaticus), was especially effective. With the addition of nucleotides, buffers, and ions, the cyclic reactions occurred in ways that convinced Mullis that he could refine the technique to generate unlimited amounts of any DNA sequence.
Beginning in the fall of 1984 and continuing into the spring of 1985, Mullis, with the aid of Cetus technicians, obtained reliable experimental data showing that they had amplified genomic DNA hundreds of thousands of times. Cetus Corporation filed for its first PCR patent on March 28, 1985, and it was accepted by the U.S. Patent Office on November 15. On September 20, 1985, a paper titled “Enzymatic Amplification of Beta-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia” was submitted to Science magazine, where it was later published. Scholars who believe that the development of the PCR technique was a team effort emphasize the many names on that paper: Randall K. Saiki, Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, and Norman Arnheim.
When Mullis later submitted his solo paper to Nature and Science, it was rejected by both of these important journals. His paper was finally published in Methods in Enzymology, the editor of which was a close friend, and it appeared in 1987 under the title “Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction.” Mullis had earlier presented his findings to an audience of molecular biologists at Cold Spring Harbor in May, 1986, when the PCR technique was enthusiastically praised. However, disheartened by what he felt was an attempt by people at Cetus to steal credit for his PCR idea, he left the company in September, 1986.
The polymerase chain reaction has become one of the most often used and extensively applied techniques in molecular biology. In 1992, when Hoffmann-La Roche
Further controversy had developed within Cetus, where several scientists and technicians challenged Mullis’s view that he deserved the lion’s share of credit for the PCR discovery. For these Cetus employees, PCR was the result of group effort rather than the idea of an isolated individual. On the other hand, the Nobel Committee reinforced Mullis’s association with PCR when they awarded him the 1993 Nobel Prize in Chemistry
In general, PCR dramatically transformed molecular biology by vastly increasing scientists’ power to identify and manipulate genetic material. In the field of medicine, PCR has been used in the diagnosis of bacterial and viral infections, in particular the detection and characterization of human immunodeficiency virus (HIV) in AIDS research. PCR has quickly become important in the molecular diagnosis of various cancers. Scientists have also used PCR to detect various hereditary diseases. According to James D. Watson, PCR has had its most important influence in the Human Genome Project,
Many other applications of the PCR technique continue to be developed, and some have suggested that it has produced or will produce a scientific revolution. However, the PCR discovery did not involve what science historian Thomas S. Kuhn described as a “paradigm shift,” because PCR’s discovery did not involve a radical conceptual shift but simply the development of a powerful new technique. PCR has certainly helped scientists to solve many puzzles of nature, but it has not elevated Mullis to the realm of such revolutionaries as Nicolaus Copernicus, Isaac Newton, and Albert Einstein.
-
Erlich, Henry A., ed. PCR Technology: Principles and Applications for DNA Amplification. New York: Stockton Press, 1989. Erlich, who was a senior scientist at Cetus during PCR’s development, introduces readers to some of the most important research and medical applications of PCR. References and index. -
Mullis, Kary B. Dancing Naked in the Mind Field. New York: Vintage Books, 2000. Autobiography of the eccentric, frank, and opinionated Nobel Prize winner. Index. -
Mullis, Kary B., François Ferré, and Richard A. Gibbs, eds. The Polymerase Chain Reaction. Boston: Birkhäuser, 1994. Articles are grouped in three sections devoted to methodology, applications, and “PCR and the world of business.” Includes an interesting foreword by James D. Watson and preface by Kary B. Mullis. References and index. -
Rabinow, Paul. Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press, 1996. Rabinow, an anthropologist, challenges Mullis’s account of the PCR discovery by exploring the “culture of biotechnology” at Cetus. He interviewed several of the scientists involved in the development of PCR, and he emphasizes the social and scientific context of PCR’s discovery. No index.
Cohen and Boyer Develop Recombinant DNA Technology
Berg, Gilbert, and Sanger Develop Techniques for Genetic Engineering
Geneticists Create Giant Mice
Cech Demonstrates That RNA Can Act as an Enzyme
Murray and Szostak Create the First Artificial Chromosome
Jeffreys Discovers the Technique of Genetic Fingerprinting
Erlich Develops DNA Fingerprinting from a Single Hair
Completion of the Sequencing of the Human Genome Is Announced