(This article assumes familiarity with the terms and components used in the PCR process.)
The history of the polymerase chain reaction (PCR) has variously been described as a classic "Eureka!" moment,Kary Mullis' Nobel Lecture, December 8, 1993 or as an example of cooperative teamwork between disparate researchers. Following is a list of events before, during, and after its development:
Prelude On April 25, 1953 James D. Watson and Francis Crick published "a radically different structure" for DNA,Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737–738 (1953). thereby founding the field of molecular genetics. Their structural model featured two strands of complementary base-paired DNA, running in opposite directions as a double helix. They concluded their report saying that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". For this insight they were awarded the Nobel Prize in 1962. Starting in the mid-1950s, Arthur Kornberg began to study the mechanism of DNA replication.(Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46. By 1957 he has identified the first DNA polymerase.Lehman, IR, Bessman MJ, Simms ES, Kornberg A "Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of Substrates and Partial Purification of an Enzyme from Escherichia coli" J. Biol. Chem. vol. 233(1) pp. 163–170 (1958). The enzyme was limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. Overall, the DNA replication process is surprisingly complex, requiring separate proteins to open the DNA helix, to keep it open, to create primers, to synthesize new DNA, to remove the primers, and to tie the pieces all together. Kornberg was awarded the Nobel Prize in 1959. In the early 1960s H. Gobind Khorana made significant advances in the elucidation of the genetic code. Afterwards, he initiated a large project to totally synthesize a functional human gene.Khorana HG et al. "Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction" J. Biol. Chem. vol. 251(3) pp. 565–70 (1976). To achieve this, Khorana pioneered many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligonucleotides were used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana was awarded the Nobel Prize for his work on the Genetic Code. In 1969 Thomas D. Brock reported the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. Thermus aquaticusBrock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bacteriol. vol. 98(1) pp. 289–297 (1969). (Taq) became a standard source of enzymes able to withstand higher temperatures than those from E. coli. In 1970 Klenow reported a modified version of DNA Polymerase I from E. coli.Klenow H and Henningsen I "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis" Proc Natl Acad Sci vol. 65 pp. 168–75 (1970). Treatment with a protease removed the 'forward' nuclease activity of this enzyme. The overall activity of the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than its degradation. By 1971 researchers in Khorana's project, concerned over their yields of DNA, began looking at "repair synthesis" – an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describePanet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213–21 (1974). employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR. Circa 1971 Kjell Kleppe, a researcher in Khorana's lab, envisioned a process very similar to PCR. At the end of a paper on the earlier technique,Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases." J. Molec. Biol. vol. 56, pp. 341–61 (1971). he described how a two-primer system might lead to replication of a specific segment of DNA: one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme.
No results are shown there, and the mention of unpublished experiments in another paper may (or may not) refer to the two-primer replication system. (These early precursors to PCR were carefully scrutinized in a patent lawsuit, and are discussed in Mullis' chapters in The Polymerase Chain Reaction (1994).Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ) Also in 1971, Cetus Corporation was founded in Berkeley, California, by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screened for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they began projects involving the new biotechnology industry, primarily the cloning and expression of human genes, but also the development of diagnostic tests for genetic mutations. In 1976 a DNA polymeraseChien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bacteriol. vol. 174 pp. 1550–1557 (1976). was isolated from T. aquaticus. It was found to retain its activity at temperatures above 75 °C. In 1977 Frederick Sanger reported a method for determining the sequence of DNA.Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463–7 (1977). The technique employed an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. For this innovation he was awarded the Nobel Prize in 1980.
By 1980 all of the components needed to perform PCR amplification were known to the scientific community. The use of DNA polymerase to extend oligonucleotide primers was a common procedure in DNA sequencing and the production of cDNA for cloning and expression. The use of DNA polymerase for nick translation was the most common method used to label DNA probes for Southern blotting.
Theme In 1979 Cetus Corporation hired Kary Mullis to synthesize oligonucleotides for various research and development projects throughout the company.Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56–65 (April 1990). These oligos were used as probes for screening cloned genes, as primers for DNA sequencing and cDNA synthesis, and as building blocks for gene construction. Originally synthesizing these oligos by hand, Mullis later evaluated early prototypes for automated synthesizers. By May 1983 Mullis synthesized oligonucleotide probes for a project at Cetus to analyze a sickle cell anemia mutation. Hearing of problems with their work, Mullis proposed an alternative technique based on Sanger's DNA sequencing method. Realizing the difficulty in making the Sanger method specific to a single location in the genome, Mullis then modified the idea to add a second primer on the opposite strand. Repeated applications of polymerase could lead to a chain reaction of replication for a specific segment of the genome – PCR. Later in 1983 Mullis began to test his idea. His first experiment did not involve thermal cycling – he hoped that the polymerase could perform continued replication on its own. Later experiments that year included repeated thermal cycling, and targeted small segments of a cloned gene. Mullis considered these experiments a success, but could not convince other researchers. In June 1984 Cetus held its annual meeting in Monterey, California. Its scientists and consultants presented their results, and considered future projects. Mullis presented a poster on the production of oligonucleotides by his laboratory, and presented some of the results from his experiments with PCR. Only Joshua Lederberg, a Cetus consultant, showed any interest. Later at the meeting, Mullis was involved in a physical altercation with another Cetus researcher over a dispute unrelated to PCR. The other scientist left the company, and Mullis was removed as head of the oligo synthesis lab.
Development In September 1984 Tom White, VP of Research at Cetus (and a close friend), pressured Mullis to take his idea to the group developing the genetic mutation assay. Together, they spent the following months designing experiments that could convincingly show that PCR is working on genomic DNA. Unfortunately, the expected amplification product was not visible in agarose gel electrophoresis, leading to confusion as to whether the reaction was specific to the targeted region. In November 1984 the amplification products were analyzed by Southern blotting, which clearly demonstrated increasing amounts of the expected 110 bp DNA product.Saiki RK et al. "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350–54 (1985). Having the first visible signal, the researchers began optimizing the process. Later, the amplified products were cloned and sequenced, showing that most of the amplified DNA was the desired target, and that the Klenow fragment then being used only rarely incorporated incorrect nucleotides during replication.
Exposition Following normal industrial practice, Mullis applied Mullis KB "Process for amplifying nucleic acid sequences." . for a patent covering the basic idea of PCR and many potential applications, and was asked by the PTO to include more results. On March 28, 1985, Mullis' development group filed an applicationMullis, KB et al. "Process for amplifying, detecting, and/or-cloning nucleic acid sequences." . focused on the analysis of the sickle cell anemia mutation via PCR and Oligomer restriction. After modification, both patents were approved on July 28, 1987.
In the spring of 1985 the development group began to apply the PCR technique to other targets. Primers and probes were designed for a variable segment of the Human leukocyte antigen DQα gene. This reaction was much more specific than that for the β-hemoglobin target – the expected PCR product is directly visible on agarose gel electrophoresis. The amplification products from various sources were also cloned and sequenced, the first determination of new alleles by PCR. At the same time the original Oligomer Restriction assay technique was replaced with the more general Allele specific oligonucleotide method.Saiki et al. "Analysis of enzymatically amplified β-globin and HLA DQα DNA with allele-specific oligonucleotide probes." Nature vol. 324 (6093) pp. 163–6 (1986).