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Polymerase chain reaction (PCR) - WWW.OLOSCIENCE.COM
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The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Developed in 1984 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize in Chemistry for his work on PCR.
Length: 278
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polymerase chain reaction
Taq polymerase is used to replicate targeted portions of the genome during a cycle of heating and cooling. The process may be repeated thirty or forty times to get the right quantity of DNA by Gautam Rangan
Length: 27
Rating: 3.70 (19 ratings)
Tags: DNA polymerase chain reaction PCR Gautam Rangan
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Kary Mullis, father of Polymerase Chain Reaction (PCR)
Kary Banks Mullis, Ph.D. (born December 28, 1944) is an American biochemist and Nobel laureate.
Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith. Mullis received the prize for his development of the Polymerase Chain Reaction (PCR), a reaction first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana that allows the amplification of specific DNA sequences.[1] The improvements provided by Mullis have made PCR a central technique in biochemistry and molecular biology. Mullis also received the Japan Prize in 1993.
Mullis was born in Lenoir, North Carolina, near the Blue Ridge Mountains,[2] on December 28, 1944. His family had a background in farming in this rural area. As a child, Mullis recalls, he was interested in observing biological organisms in the countryside.[1] He grew up in Columbia, South Carolina,[1] where he attended Dreher High School.
Mullis earned a Bachelor of Science degree in chemistry[2] from the Georgia Institute of Technology in Atlanta in 1966 and received a Ph.D. in biochemistry from the University of California, Berkeley in 1973; his research focused on synthesis and structure of proteins.[1] Following his graduation, Mullis became a postdoctoral fellow in paediatric cardiology at the University of Kansas Medical School, going on to complete two years of postdoctoral work in pharmaceutical chemistry at the University of California, San Francisco.
After receiving his PhD, Mullis left science to write fiction, then managed a bakery for two years.[3] Mullis returned to science at the encouragement of friend Thomas White, who later got Mullis a job with the biotechnology company Cetus Corporation of Emeryville, California.[3][1] Mullis worked as a DNA chemist at Cetus for seven years; it was there, in 1983, that Mullis invented his prize-winning improvements to the polymerase chain reaction.[4] After leaving Cetus in 1986, Mullis served as director of molecular biology for Xytronyx, Inc. for two years. Mullis has consulted on nucleic acid chemistry for multiple corporations.[3]
In 1992, Mullis founded a business with the intent to sell pieces of jewelry containing the amplified DNA of deceased famous people like Elvis Presley and Marilyn Monroe.[5][6]
Mullis was not the first to propose the ideas behind PCR. The main principles were described in 1971 by 1968 Nobel Prize laureate H. Gobind Khorana and Kjell Kleppe, a Norwegian scientist. Kleppe and Khorana released a 20-page research paper on PCR in the 1971 Journal of Molecular Biology. As early as June 18, 1969, Kleppe had presented his work at the Gordon Conference in New Hampshire. Using repair replication (the principle of PCR), he duplicated and then quadrupled a small synthetic molecule with the help of two primers and DNA-polymerase. Among the attendees[7] was Stuart Linn, who then used Kleppe's material in his own teachings to his students, including Mullis.
The suggestion that Mullis was solely responsible for the idea of using Taq polymerase in the PCR process has been refuted by his co-workers at the time.[citation needed] However, other scientists have said that "the full potential [of PCR] was not realized" until Mullis' work in 1983,[8] and at least one book has reported that Mullis' colleagues failed to see the potential of the technique when he presented it to them.[5] As a result, some controversy surrounds the balance of credit that should be given to Mullis versus the team at Cetus.[3] In practice, credit has accrued to both the inventor and the company (although not its individual workers) in the form of a Nobel Prize and a $10,000 Cetus bonus for Mullis and $300 million for Cetus when the company sold the patent to Roche Molecular Systems.
The anthropologist Paul Rabinow wrote a book on the history of the PCR method in 1996 (entitled 'Making PCR') in which he questioned whether or not Mullis "invented" PCR or "merely" came up with the concept of it. Rabinow, a Foucault scholar interested in issues of the production of knowledge, used the topic to argue against the idea that scientific discovery is the product of individual work, writing, "Committees and science journalists like the idea of associating a unique idea with a unique person, the lone genius. PCR is, in fact, one of the classic examples of teamwork."[9]
In a Q&A interview published in the September, 1994, issue of California Monthly, Mullis said, "Back in the 1960s and early '70s I took plenty of LSD. A lot of people were doing that in Berkeley back then. And I found it to be a mind-opening experience. It was certainly much more important than any courses I ever took."[15] During a symposium held for centenarian Albert Hofmann, "Hofmann revealed that he was told by Nobel-prize-winning chemist Kary Mullis that LSD had helped him develop the polymerase chain reaction that helps amplify specific DNA sequences."[16]
Length: 254
Rating: 5.00 (1 ratings)
Tags: biology evolution genes biological theory self molecular DNA
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DNA Test Methods - The Polymerase Chain Reaction
View the full Interactive Tutorial at:
http://www.phgfoundation.org/tutorials/dna/4.html
The polymerase chain reaction, or PCR, is a crucial method for exponentially increasing the amount of a specific DNA sequence.
PCR is a cyclic process controlled by the temperature of the reaction mixture. First, the temperature is raised to 95°C (205°F), causing the template DNA to separate (denature).
The temperature is then decreased to around 50°C (122°F), allowing short primer pieces of DNA to hybridise to each strand of the template at opposite ends of the sequence to be amplified.
The thermostable enzyme Taq (a DNA polymerase that is active at high temperatures) then binds to and extends the primers at an intermediate temperature of 72°C (162°F), so that two new double-stranded copies of the template are made.
This cyclic process of separating DNA strands, copying and reannealing the daughter strands is repeated multiple times to increase the numbers of DNA product.
Length: 60
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Tags: phgfoundation interactive tutorials dna test methods
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Phire DNA polymerase
Phire DNA polymerase
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Negative A - Polymerase Chain Reaction
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Polymerase Chain Reaction (PCR) Song
an hymn to the technique widely used in molecular biology
Length: 134
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