History of polymerase chain reaction

Last updated October 09, 2023

(This article assumes familiarity with the terms and components used in the PCR process.)

DNA Polymerase I (PDB) DNAPolymeraseI.jpg — DNA Polymerase I (PDB)

Molecular mechanism of PCR PCR.svg — Molecular mechanism of PCR

The history of the polymerase chain reaction (PCR) has variously been described as a classic "Eureka!" moment,^[1] or as an example of cooperative teamwork between disparate researchers.^[2] 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,^[3] 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.^[4] By 1957 he has identified the first DNA polymerase.^[5] 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.^[6] 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 aquaticus ^[7] (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.^[8] 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 describe^[9] 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,^[10] he described how a two-primer system might lead to replication of a specific segment of DNA:^[10]

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^[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 The Polymerase Chain Reaction (1994).^[11])

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 polymerase ^[12] 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.^[13] 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.^[14] 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.^[1]
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.^[14] 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^[2] 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.^[2] Only Joshua Lederberg, a Cetus consultant, showed any interest.^[14] Later at the meeting, Mullis was involved in a physical altercation with another Cetus researcher over a dispute unrelated to PCR.^[2] 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,^[15] leading to confusion as to whether the reaction had any specificity to the targeted region.
In November 1984^[2] the amplification products were analyzed by Southern blotting, which clearly demonstrating increasing amount of the expected 110 bp DNA product.^[16] Having the first visible signal, the researchers began optimizing the process. Later, the amplified products were cloned and sequenced, showing that only a small fraction of the amplified DNA is the desired target, and that the Klenow fragment then being used only rarely incorporates incorrect nucleotides during replication.^[15]

Exposition

Following normal industrial practice, Mullis applied ^[17] 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 application^[18] 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^[15] 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.^[15] At the same time the original Oligomer Restriction assay technique was replaced with the more general Allele specific oligonucleotide method.^[19]
Also early in 1985, the group began using a thermostable DNA polymerase (the enzyme used in the original reaction is destroyed at each heating step). At the time^[1] only two had been described, from Taq and Bst. The report on Taq polymerase^[12] was more detailed, so it was chosen for testing. The Bst polymerase was later found to be unsuitable for PCR^{[ citation needed ]}. That summer Mullis attempted to isolate the enzyme, and a group outside of Cetus was contracted to make it, all without success. In the Fall of 1985 Susanne Stoffel and David Gelfand at Cetus succeed in making the polymerase, and it was immediately found by Randy Saiki to support the PCR process.
With patents submitted, work proceeded to report PCR to the general scientific community. An abstract for an American Society of Human Genetics meeting in Salt Lake City was submitted in April 1985, and the first announcement of PCR was made there by Saiki in October.^[20] Two publications were planned – an 'idea' paper from Mullis, and an 'application' paper from the entire development group. Mullis submitted his manuscript to the journal Nature, which rejected it for not including results. The other paper, mainly describing the OR analysis assay, was submitted to Science on September 20, 1985, and was accepted in November. After the rejection of Mullis' report in December, details on the PCR process were hastily added to the second paper, which appears on December 20, 1985.^[16]
In May 1986 Mullis presented PCR at the Cold Spring Harbor Symposium,^[21] and published a modified version of his original 'idea' manuscript much later.^[22] The first non-Cetus report using PCR was submitted on September 5, 1986,^[23] indicating how quickly other laboratories began implementing the technique. The Cetus development group published their detailed sequence analysis of PCR products on September 8, 1986,^[15] and their use of ASO probes on November 13, 1986.^[19]
The use of Taq polymerase in PCR was announced by Henry Erlich at a meeting in Berlin on September 20, 1986, submitted for publication in October 1987, and was published early the next year'.^[24] The patent for PCR with Taq polymerase was filed on June 17, 1987, and was issued on October 23, 1990.^[25]

Variation

In December 1985 a joint venture between Cetus and Perkin-Elmer was established to develop instruments and reagents for PCR. Complex thermal cyclers were constructed to perform the Klenow-based amplifications, but never marketed. Simpler machines for Taq-based PCR were developed, and on November 19, 1987, a press release announces the commercial availability of the "PCR-1000 Thermal Cycler" and "AmpliTaq DNA Polymerase". In the spring of 1985 John Sninsky at Cetus began to use PCR for the difficult task of measuring the amount of HIV circulating in blood. A viable test was announced on April 11, 1986, and published in May 1987.^[26] Donated blood could then be screened for the virus, and the effect of antiviral drugs directly monitored.

In 1985 Norm Arnheim, also a member of the development team, concluded his sabbatical at Cetus and assumed an academic position at University of Southern California. He began to investigate the use of PCR to amplify samples containing just a single copy of the target sequence. By 1989 his lab developed multiplex-PCR on single sperm to directly analyze the products of meiotic recombination.^[27] These single-copy amplifications, which had first been run during the characterization of Taq polymerase,^[24] became vital to the study of ancient DNA, as well as the genetic typing of preimplanted embryos.

In 1986 Edward Blake, a forensics scientist working in the Cetus building, collaborated with Henry Erlich a researcher at Cetus, to apply PCR to the analysis of criminal evidence. A panel of DNA samples from old cases was collected and coded, and was analyzed blind by Saiki using the HLA DQα assay. When the code was broken, all of the evidence and perpetrators matched. Blake and Erlich's group used the technique almost immediately in Pennsylvania v. Pestinikas,^[28] the first use of PCR in a criminal case. This DQα test is developed by Cetus as one of their "Ampli-Type" kits, and became part of early protocols for the testing of forensic evidence, such as in the O. J. Simpson murder case.

By 1989 Alec Jeffreys, who had earlier developed and applied the first DNA Fingerprinting tests, used PCR to increase their sensitivity.^[29] With further modification, the amplification of highly polymorphic Variable number tandem repeat (VNTR) loci became the standard protocol for National DNA Databases such as Combined DNA Index System (CODIS).

In 1987 Russ Higuchi succeeded in amplifying DNA from a human hair.^[30] This work expanded to develop methods for the amplification of DNA from highly degraded samples, such as from ancient DNA and in forensic evidence.

Coda

On December 22, 1989, the journal Science awarded Taq Polymerase (and PCR) its first "Molecule of the Year". The 'Taq PCR' paper^[24] became for several years the most cited publication in biology.
After the publication of the first PCR paper,^[16] the United States Government sent a stern letter to Randy Saiki, admonishing him for publishing a report on "chain reactions" without the required prior review and approval by the U.S. Department of Energy. Cetus responded, explaining the differences between PCR and the atomic bomb.^{[ citation needed ]}
On July 23, 1991, Cetus announced that its sale to the neighboring biotechnology company Chiron. As part of the sale, rights to the PCR patents were sold for US$300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers moved to the Roche subsidiary, Roche Molecular Systems.
On October 13, 1993, Kary Mullis, who had left Cetus in 1986, was awarded the Nobel Prize in Chemistry. On the morning of his acceptance speech,^[1] he was nearly arrested by Swedish authorities for the "inappropriate use of a laser pointer".^[31]

Related Research Articles

<span class="mw-page-title-main">Kary Mullis</span> American biochemist (1944–2019)

Kary Banks Mullis was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and was awarded the Japan Prize in the same year. PCR became a central technique in biochemistry and molecular biology, described by The New York Times as "highly original and significant, virtually dividing biology into the two epochs of before PCR and after PCR."

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

DNA synthesis is the natural or artificial creation of deoxyribonucleic acid (DNA) molecules. DNA is a macromolecule made up of nucleotide units, which are linked by covalent bonds and hydrogen bonds, in a repeating structure. DNA synthesis occurs when these nucleotide units are joined to form DNA; this can occur artificially or naturally. Nucleotide units are made up of a nitrogenous base, pentose sugar (deoxyribose) and phosphate group. Each unit is joined when a covalent bond forms between its phosphate group and the pentose sugar of the next nucleotide, forming a sugar-phosphate backbone. DNA is a complementary, double stranded structure as specific base pairing occurs naturally when hydrogen bonds form between the nucleotide bases.

<span class="mw-page-title-main">DNA polymerase I</span> Family of enzymes

DNA polymerase I is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase. It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.

Thermus aquaticus is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the Deinococcota phylum. It is the source of the heat-resistant enzyme Taq DNA polymerase, one of the most important enzymes in molecular biology because of its use in the polymerase chain reaction (PCR) DNA amplification technique.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification that takes place at a constant temperature.

<span class="mw-page-title-main">Klenow fragment</span>

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions to copy the organism's DNA during cell division. In the laboratory setting, Pfu is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step.

The overlap extension polymerase chain reaction is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used assemble multiple smaller double stranded DNA fragments into a larger DNA sequence. OE-PCR is widely used to insert mutations at specific points in a sequence or to assemble custom DNA sequence from smaller DNA fragments into a larger polynucleotide.

TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications.

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler dot blot assay. It is a common tool used in genetic testing, forensics, and molecular biology research.

The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.

Polymerase cycling assembly is a method for the assembly of large DNA oligonucleotides from shorter fragments. The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process. It thus allows for the production of synthetic genes and even entire synthetic genomes.

<span class="mw-page-title-main">Oligomer restriction</span>

Oligomer Restriction is a procedure to detect an altered DNA sequence in a genome. A labeled oligonucleotide probe is hybridized to a target DNA, and then treated with a restriction enzyme. If the probe exactly matches the target, the restriction enzyme will cleave the probe, changing its size. If, however, the target DNA does not exactly match the probe, the restriction enzyme will have no effect on the length of the probe. The OR technique, now rarely performed, was closely associated with the development of the popular polymerase chain reaction (PCR) method.

The versatility of polymerase chain reaction (PCR) has led to modifications of the basic protocol being used in a large number of variant techniques designed for various purposes. This article summarizes many of the most common variations currently or formerly used in molecular biology laboratories; familiarity with the fundamental premise by which PCR works and corresponding terms and concepts is necessary for understanding these variant techniques.

Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room temperatures. Many variations and modifications of the PCR procedure have been developed in order to achieve higher yields; hot start PCR is one of them. Hot start PCR follows the same principles as the conventional PCR - in that it uses DNA polymerase to synthesise DNA from a single stranded template. However, it utilizes additional heating and separation methods, such as inactivating or inhibiting the binding of Taq polymerase and late addition of Taq polymerase, to increase product yield as well as provide a higher specificity and sensitivity. Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation. Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures, use of modified deoxyribonucleotide triphosphates (dNTPs), and the physical addition of one of the essential reagents after denaturation.

References

1 2 3 4 Kary Mullis' Nobel Lecture, December 8, 1993
1 2 3 4 5 Rabinow, Paul (1996). Making PCR : a story of biotechnology . University of Chicago Press. ISBN 978-0-226-70146-2.^{[ page needed ]}
↑ Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737–738 (1953).
↑ (Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46.
↑ 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).
↑ 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).
↑ Brock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bacteriol. vol. 98(1) pp. 289–297 (1969).
↑ 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).
1 2 Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213–21 (1974).
1 2 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).
↑ Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ISBN 0-8176-3750-8
1 2 Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bacteriol. vol. 174 pp. 1550–1557 (1976).
↑ Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463–7 (1977).
1 2 3 Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56–65 (April 1990).
1 2 3 4 5 Scharf et al. "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences" Science vol. 233, pp. 1076–78 (1986).
1 2 3 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).
↑ Mullis KB "Process for amplifying nucleic acid sequences." U.S. Patent 4,683,202 .
↑ Mullis, KB et al. "Process for amplifying, detecting, and/or-cloning nucleic acid sequences." U.S. Patent 4,683,195 .
1 2 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).
↑ Saiki, R et al. "A Novel Method for the Prenatal Diagnosis of Sickle Cell Anemia" Amer. Soc. Human Genetics, Oct. 9–13, 1985.
↑ Mullis KB et al. "Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction." Cold Spring Harb. Symp. Quant. Biol. vol. 51 pp. 263–73 (1986).
↑ Mullis KB and Faloona FA "Specific Synthesis of DNA in vitro via a Polymerase-Catalyzed Chain Reaction." Methods in Enzymology vol. 155(F) pp. 335–50 (1987).
↑ Verlaan-de Vries M et al. "A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides." Gene vol. 50(1–3) pp. 313–20 (1986).
1 2 3 Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487–91 (1988).
↑ Mullis, KB et al. "Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme." U.S. Patent 4,965,188 .
↑ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690–4 (1987).
↑ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21–32 (1989).
↑ "Forensic Science Timeline (PDF)" (PDF). Archived from the original (PDF) on 2008-07-09. Retrieved 2008-04-12.
↑ Jeffreys A et al. "Amplification of human minisatellites." Nucleic Acids Research vol. 23 pp. 10953-71 (1988).
↑ Higuchi R et al. "DNA typing from single hairs." Nature vol. 332(6164) pp. 543–6 (1988).
↑ Mullis KB "Dancing Naked in the Mind Field" Pantheon Books (1998) ISBN 0-679-44255-3

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[MullisLecture-1] 1 2 3 4 Kary Mullis' Nobel Lecture, December 8, 1993

[Rabinow-2] 1 2 3 4 5 Rabinow, Paul (1996). Making PCR : a story of biotechnology . University of Chicago Press. ISBN 978-0-226-70146-2.^{[ page needed ]}

[3] Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737–738 (1953).

[4] (Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46.

[5] 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).

[6] 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).

[7] Brock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bacteriol. vol. 98(1) pp. 289–297 (1969).

[8] 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).

[Khorana1-9] 1 2 Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213–21 (1974).

[Kleppe-10] 1 2 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).

[11] Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ISBN 0-8176-3750-8

[Chien-12] 1 2 Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bacteriol. vol. 174 pp. 1550–1557 (1976).

[13] Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463–7 (1977).

[Mullis-1-14] 1 2 3 Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56–65 (April 1990).

[Scharf-15] 1 2 3 4 5 Scharf et al. "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences" Science vol. 233, pp. 1076–78 (1986).

[Saiki1-16] 1 2 3 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).

[17] Mullis KB "Process for amplifying nucleic acid sequences." U.S. Patent 4,683,202 .

[18] Mullis, KB et al. "Process for amplifying, detecting, and/or-cloning nucleic acid sequences." U.S. Patent 4,683,195 .

[Saiki2-19] 1 2 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).

[20] Saiki, R et al. "A Novel Method for the Prenatal Diagnosis of Sickle Cell Anemia" Amer. Soc. Human Genetics, Oct. 9–13, 1985.

[21] Mullis KB et al. "Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction." Cold Spring Harb. Symp. Quant. Biol. vol. 51 pp. 263–73 (1986).

[22] Mullis KB and Faloona FA "Specific Synthesis of DNA in vitro via a Polymerase-Catalyzed Chain Reaction." Methods in Enzymology vol. 155(F) pp. 335–50 (1987).

[23] Verlaan-de Vries M et al. "A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides." Gene vol. 50(1–3) pp. 313–20 (1986).

[Saiki3-24] 1 2 3 Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487–91 (1988).

[25] Mullis, KB et al. "Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme." U.S. Patent 4,965,188 .

[26] Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690–4 (1987).

[27] Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21–32 (1989).

[28] "Forensic Science Timeline (PDF)" (PDF). Archived from the original (PDF) on 2008-07-09. Retrieved 2008-04-12.

[29] Jeffreys A et al. "Amplification of human minisatellites." Nucleic Acids Research vol. 23 pp. 10953-71 (1988).

[30] Higuchi R et al. "DNA typing from single hairs." Nature vol. 332(6164) pp. 543–6 (1988).

[31] Mullis KB "Dancing Naked in the Mind Field" Pantheon Books (1998) ISBN 0-679-44255-3

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