History of Polymerase chain reaction (PCR)--(2)

Exposition

As per normal industrial practice, the results are first used to apply for patents. Mullis prepares an application[17] for the basic idea of PCR and many potential applications, and is asked by the PTO to include more results. On March 28, 1985 the entire development group (including Mullis) files an application[18] that is more focused on the analysis of the SCA mutation via PCR and OR. After modification, both patents are approved on July 28, 1987.


In the spring of 1985 the development group begins to apply PCR to other targets. Primers and probes are designed for a variable segment of the HLA DQα gene. This reaction turns out to be 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 are also cloned and sequenced, the first determination of new alleles by PCR[15]. At this same time the original OR assay technique is replaced with the more general ASO method[19].

Also early in 1985, the group turns its attention to the use of a thermostable DNA polymerase (the enzyme used in the original reaction is destroyed at each heating step). A literature search[1] reveals that only two have been described, from Taq and Bst. The report on Taq polymerase[12] is more detailed, so it is chosen for testing. A fortuitous decision - the Bst polymerase is later found to be unsuitable for PCR[citation needed]. That summer Mullis tries twice to isolate the enzyme, and a group outside of Cetus is also 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 is immediately found by Randy Saiki to support the PCR process.

With patents submitted, work proceeds for reporting PCR to the general scientific community. An abstract for a meeting in Salt Lake City is submitted in April 1985, and the first announcement of PCR is made there by Saiki in October[20]. Two publications are planned - an 'idea' paper from Mullis, and an 'application' paper from the entire development group. Mullis submits his manuscript to the journal Nature, which rejects it for not including results. The other paper, mainly describing the OR analysis assay, is submitted to Science on September 20, 1985 and is accepted in November. After the rejection of Mullis' report in December, details on the PCR process are hastily added to the second paper, which appears on December 20, 1985[16].

In May of 1986 Mullis presents PCR at the Cold Spring Harbor Symposium[21], and publishes a modified version of his original 'idea' manuscript much later[22]. The first non-Cetus report using PCR is submitted on September 5, 1986[23], indicating how quickly other laboratories are implementing the technique. The Cetus development group publishes 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 is announced by Henry Erlich at a meeting in Berlin on September 20, 1986, is submitted for publication in October of 1987, and is published early the next year'[24]. The patent for PCR with Taq polymerase is filed on June 17, 1987, and is issued on October 23, 1990[25].

Variation

In December 1985 a joint venture between Cetus and Perkin-Elmer is established to develop instruments and reagents for PCR. Complex Thermal Cyclers are constructed to perform the Klenow-based amplifications, but are never marketed. Simpler machines for Taq-based PCR are 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 begins to apply PCR to the difficult task of quantitating the amount of HIV circulating in blood. A viable test is announced on April 11, 1986, and is published in May 1987[26] . Donated blood can now be screened for the virus, and the effect of antiviral drugs can be directly monitored.

In 1985 Norm Arnheim, also a member of the development team, concludes his sabbatical at Cetus and gets a real job at USC. He begins to investigate the use of PCR to amplifiy samples containing just a single copy of the target sequence. By 1989 his lab runs mutiplex-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], become 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, collaborates with Bruce Budowle (of the FBI) and Cetus researchers to apply PCR to the analysis of criminal evidence. A panel of DNA samples from old cases is collected and coded, and is analyzed blind by Saiki using the HLA DQα assay. When the code is broken, all of the evidence and perpetrators match. Blake uses 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 goes on to become part of early protocols for the testing of forensic evidence.

By 1989 Alec Jeffreys, who had earlier developed and applied the first DNA Fingerprinting tests, uses PCR to increase their sensitivity[29]. With further modification, the amplification of highly polymorphic VNTR loci will become the standard protocol for National DNA Databases such as CODIS. The guilty go to jail, and the ability of PCR to restest old evidence begins to set the innocent free.

In 1987 Russ Higuchi succeeds in amplifying DNA from a human hair[30]. This work expands to develop methods to amplify DNA from highly degraded samples, such as from Ancient DNA and in forensic evidence. On January 30, 1989 an episode of Star Trek: The Next Generation airs. The ship's doctor is being rapidly aged by a virus attacking her DNA, and is cured when her pre-infection DNA is isolated from a hair found in her cabin. PCR has entered the mainstream media.

Coda

On December 22, 1989 the journal Science awards Taq Polymerase (and PCR) its first "Molecule of the Year". The 'Taq PCR' paper[24] goes on to become (for several years) the most cited publication in biology.

After the publication of the first PCR paper[16], the United States Government sends 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 writes back, explaining the differences between PCR and the atomic bomb.

On July 23, 1991 Cetus announces that it will be sold to its neighboring biotechnology company Chiron. As part of the sale, rights to the PCR patents are sold for USD $300 million to Hoffman-La Roche (who in 1989 had bought limited rights to PCR). Many of the Cetus PCR researchers move to a new subsidiary, Roche Molecular Systems.

On October 13, 1993 Kary Mullis, who had left Cetus in 1986, is awarded the Nobel Prize in Chemistry. On the morning of his acceptance speech[1], he is nearly arrested by Swedish authorities for the "inappropriate use of a laser pointer"[31].

References

^ a b c d Kary Mullis' Nobel Lecture, December 8, 1993
^ a b c d e Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996) ISBN 0-226-70147-6
^ Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737-738 (1953). [1]
^ (Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46. [2]
^ 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. Bact. 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).
^ a b Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213-21 (1974).
^ a b 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 0-817-63750-8
^ a b Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bact. 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).
^ a b c Mullis KB "The Unusual Origins of the Polymerase Chain Reaction" Scientific American, vol. 262, pp. 56-65 (April 1990).
^ a b c d e Scharf et al. "Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences" Science vol. 233, pp. 1076-78 (1986).
^ a b c 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.
^ a b 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 Harbor 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).
^ a b c 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).
^ 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