Editing Polymerase chain reaction (section)

==History==
{{Main|History of polymerase chain reaction}}
[[File:Primers RevComp.svg|thumb|Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation that allowed the development of PCR.]]

The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of [[Yellowstone National Park|Yellowstone]]'s Mushroom Spring.<ref>{{Cite web|title=Key ingredient in coronavirus tests comes from Yellowstone's lakes|url=https://www.nationalgeographic.com/science/2020/03/key-ingredient-in-coronavirus-tests-comes-from-yellowstone/|archive-url=https://web.archive.org/web/20200331153001/https://www.nationalgeographic.com/science/2020/03/key-ingredient-in-coronavirus-tests-comes-from-yellowstone/|url-status=dead|archive-date=31 March 2020|date=2020-03-31|website=Science|language=en|access-date=2020-05-13}}</ref>

A 1971 paper in the ''[[Journal of Molecular Biology]]'' by [[Kjell Kleppe]] and co-workers in the laboratory of [[Har Gobind Khorana|H. Gobind Khorana]] first described a method of using an enzymatic assay to replicate a short DNA template with primers ''in vitro''.<ref>{{cite journal | vauthors = Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG | title = Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases | journal = Journal of Molecular Biology | volume = 56 | issue = 2 | pages = 341–61 | date = March 1971 | pmid = 4927950 | doi = 10.1016/0022-2836(71)90469-4 }}</ref> However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to [[Kary Mullis]].<ref>{{cite book|last=Rabinow|first=Paul|author-link=Paul Rabinow|year=1996|title=Making PCR: A Story of Biotechnology|publisher=University of Chicago Press|location=Chicago|isbn=978-0-226-70146-2|url-access=registration|url=https://archive.org/details/makingpcrstoryof00rabi/mode/1up}}</ref>{{Page needed|date=December 2021|reason=Rabinow in fact distinguishes between the "invention" of PCR and the "concept" of PCR in the introduction}}

[[File:Baby Blue - a prototype polymerase chain reaction (PCR), c 1986. (9663810586).jpg|thumb|right|"Baby Blue", a 1986 prototype machine for doing PCR]]
When Mullis developed the PCR in 1983, he was working in [[Emeryville, California|Emeryville]], California for [[Cetus Corporation]], one of the first [[biotechnology]] companies, where he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived the idea for PCR while cruising along the [[California State Route 1|Pacific Coast Highway]] one night in his car.<ref name=Mullis>{{cite book|last=Mullis|first=Kary|author-link=Kary Mullis|year=1998|title=Dancing Naked in the Mind Field|publisher=Pantheon Books|location=New York|isbn=978-0-679-44255-4|url-access=registration|url=https://archive.org/details/dancingnakedinmi00mull}}</ref> He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In ''[[Scientific American]]'', Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."<ref>{{cite journal | vauthors = Mullis KB | title = The unusual origin of the polymerase chain reaction | journal = Scientific American | volume = 262 | issue = 4 | pages = 56–61, 64–65 | date = April 1990 | pmid = 2315679 | doi = 10.1038/scientificamerican0490-56 | bibcode = 1990SciAm.262d..56M }}</ref> DNA fingerprinting was first used for [[paternity testing]] in 1988.<ref>{{cite journal | vauthors = Patidar M, Agrawal S, Parveen F, Khare P | title = Molecular insights of saliva in solving paternity dispute | journal = Journal of Forensic Dental Sciences | volume = 7 | issue = 1 | pages = 76–79 | date = 2015 | pmid = 25709326 | pmc = 4330625 | doi = 10.4103/0975-1475.150325 | doi-access = free }}</ref>

Mullis has credited his use of [[LSD]] as integral to his development of PCR: "Would I have invented PCR if I hadn't taken LSD?  I seriously doubt it.  I could sit on a DNA molecule and watch the polymers go by.  I learnt that partly on psychedelic drugs."<ref>{{cite journal | vauthors = Nichols D, Barker E| title = Psychedelics | journal = Pharmacological Reviews | volume = 68 | issue = 2 | pages = 264–355 | date = 2016 | pmid = 26841800 | pmc = 4813425 | doi = 10.1124/pr.115.011478 }}</ref>

Mullis and biochemist [[Michael Smith (chemist)|Michael Smith]], who had developed other essential ways of manipulating DNA,<ref name="NobelPrize">{{Cite web|url=https://www.nobelprize.org/prizes/chemistry/1993/press-release/|title=The Nobel Prize in Chemistry 1993|website=NobelPrize.org}}</ref> were jointly awarded the [[Nobel Prize in Chemistry]] in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice.<ref name="Kary Mullis Nobel Lecture">{{Cite web|url=https://www.nobelprize.org/prizes/chemistry/1993/mullis/lecture/|title=The Nobel Prize in Chemistry 1993|website=NobelPrize.org}}</ref> Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR)—was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.<ref name="breakthrough">{{cite web|title=Citations for Chemical Breakthrough Awards 2017 Awardees|url=http://www.scs.illinois.edu/~mainzv/HIST/awards/CCB-2017_Awardees.php|website=Division of the History of Chemistry|access-date=12 March 2018}}</ref><ref name="Saiki1"/>

At the core of the PCR method is the use of a suitable [[DNA polymerase]] able to withstand the high temperatures of >{{convert|90|°C|°F|abbr=on}} required for separation of the two DNA strands in the [[DNA double helix]] after each [[DNA replication|replication]] cycle. The DNA polymerases initially employed for [[in vitro]] experiments presaging PCR were unable to withstand these high temperatures.<ref name="Saiki1"/> So the early procedures for DNA replication were very inefficient and time-consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.

The discovery in 1976 of [[Taq polymerase|''Taq'' polymerase]]—a DNA polymerase purified from the [[Thermophile|thermophilic bacterium]], ''[[Thermus aquaticus]]'', which naturally lives in hot ({{convert|50|to|80|C|F}}) environments<ref name="Chien et al."/> such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from ''T. aquaticus'' is stable at high temperatures remaining active even after DNA denaturation,<ref name="Lawyer et al."/> thus obviating the need to add new DNA polymerase after each cycle.<ref name="Saiki2"/> This allowed an automated thermocycler-based process for DNA amplification.

===Patent disputes===
The PCR technique was patented by [[Kary Mullis]] and assigned to [[Cetus Corporation]], where Mullis worked when he invented the technique in 1983. The ''Taq'' polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit<ref>{{cite web | url=https://dukespace.lib.duke.edu/dspace/handle/10161/8127/recent-submissions?offset=0 | title=Recently added }}</ref> brought by [[DuPont]]. The Swiss pharmaceutical company [[Hoffmann-La Roche]] purchased the rights to the patents in 1992. The last of the commercial PCR patents expired in 2017.<ref>{{cite journal|title=The effects of business practices, licensing, and intellectual property on development and dissemination of the polymerase chain reaction: case study|date=3 July 2006|pmc=1523369|last1=Fore|first1=J. Jr.|last2=Wiechers|first2=I. R.|last3=Cook-Deegan|first3=R.|journal=Journal of Biomedical Discovery and Collaboration|volume=1|page=7|doi=10.1186/1747-5333-1-7|pmid=16817955 |doi-access=free }}</ref>

A related patent battle over the ''Taq'' polymerase enzyme is still ongoing{{as of?|date=April 2022}} in several jurisdictions around the world between Roche and [[Promega]]. The legal arguments have extended beyond the lives of the original PCR and ''Taq'' polymerase patents, which expired on 28 March 2005.<ref>{{cite journal|url=https://www.genengnews.com/magazine/49/advice-on-how-to-survive-the-taq-wars/|title=Advice on How to Survive the Taq Wars|journal=GEN Genetic Engineering News – Biobusiness Channel|date=1 May 2006|volume=26|issue=9|access-date=24 April 2019|archive-date=26 March 2020|archive-url=https://web.archive.org/web/20200326160735/https://www.genengnews.com/magazine/49/advice-on-how-to-survive-the-taq-wars/|url-status=dead}}</ref>