History of Polymerase chain reaction (PCR)--(1)

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The history of the Polymerase Chain Reaction (or PCR) has variously been described as a classic "Eureka!" moment[1], or as an example of cooperative teamwork between disparate researchers[2]. A list of some of the events before, during, and after its development:

Prelude

On April 25, 1953 James D. Watson and Francis Crick publish "a radically different structure" for DNA[3], thereby founding the field of Molecular Genetics. Their structure involves two strands of complementary base-paired DNA, running in opposite directions as a double helix. They conclude 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". They are awarded the Nobel Prize in 1962.

Starting in the mid 1950s, Arthur Kornberg begins to study the mechanism of DNA replication[4]. By 1957 he has identified the first DNA polymerase[5]. The enzyme is surprisingly limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. However, the overall 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. He is awarded the Nobel Prize in 1959.

In the early 1960s H. Gobind Khorana participates in the discovery of the Genetic Code. Afterwards, he initiates a large project to totally synthesize a functional human gene[6]. To achieve this, he pioneers many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligos are used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana is awarded the Nobel Prize for his work on the Genetic Code.

In 1969 Thomas Brock reports the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. Naming it Thermus aquaticus[7] (Taq), it goes on to become a standard source of enzymes able to withstand higher temperatures than those from E. Coli.

In 1970 a modified version of DNA Polymerase I from E. coli is reported[8]. Treatment with a protease removes 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, begin 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 describe[9] employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR.

Also by 1971 Kjell Kleppe, a researcher in Khorana's lab, envisions a process very similar to PCR. At the end of a paper on the earlier technique[10], he describes 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." [10]

No results are shown there, and the mention of unpublished experiments in another paper[9] 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 [11].)

Also in 1971, Cetus Corporation is founded in Berkeley, California by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screens for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they take up 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 polymerase[12] is isolated from T. aquaticus. It is found to retain its activity at temperatures above 75°C.

In 1977 Frederick Sanger reports a method for determining the sequence of DNA[13]. The technique involves an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. He is awarded the Nobel Prize in 1980.

Thus, 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 hires Kary Mullis to synthesize oligonucleotides for various research and development projects throughout the company[14]. These oligos are 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 evaluates early prototypes for automated synthesizers[1].

By May 1983 Mullis has synthesized oligo probes for a project at Cetus attempting to analyze a mutation for a human genetic disease. Hearing of problems with their work, Mullis envisions an alternative technique based on Sanger's DNA sequencing method[14]. Realizing the difficulty in making that method specific to a single location in the genome, Mullis considers adding a second primer on the opposite strand. He then generalizes the idea, and realizes that repeated applications of polymerase could lead to a chain reaction of replication for a specific segment of the genome - PCR.

Later in 1983 Mullis begins to test his idea. His first experiment[2] does not involve thermal cycling - he hopes that the polymerase can perform continued replication on its own. Later experiments that year do involve repeated thermal cycling, and target small segments of a cloned gene. Mullis considers these experiments a success, but is unable to convince other researchers.

In June 1984 Cetus holds its annual meeting in Monterey, California. Its scientists and consultants present their results, and consider future projects. Mullis presents a poster on the production of oligonucleotides by his laboratory, and shows some of the results from his experiments with PCR[2]. Only Joshua Lederberg, a Cetus consultant, shows any interest[14]. Later at the meeting, Mullis is involved in a physical altercation with another Cetus researcher, over a dispute unrelated to PCR[2]. The other scientist soon leaves the company, and Mullis is removed as head of the oligo synthesis lab. The days of his continued employment at Cetus may be numbered.

Development

In September of 1984 Tom White, VP of Research at Cetus (and a close friend), pressures Mullis to take his idea to the group developing the genetic mutation assay. Together, they spend the following months designing experiments that could convincingly show that PCR is working on genomic DNA. Unfortunately, the expected amplification product is not visible in agarose gel electrophoresis[15], leading to confusion as to whether the reaction has any specificity to the targeted region.

In November of 1984[2] the amplification products are analyzed by Southern blotting, which clearly shows an increasing amount of the expected 110 bp DNA product[16]. Having the first visible signal, the researchers are able to begin finding optimum conditions for the reaction. Later, the amplified products are cloned and sequenced, showing that only a small fraction of the amplified DNA is the desired target, and that the polymerase then being used only rarely incorporates incorrect nucleotides during replication[15].