09x_PCR wikiped extras_120309

09x_PCR wikiped extras_120309 - POLYMERASECHAINREACTION

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POLYMERASE CHAIN REACTION (PCR - in vitro DNA Amplification) LABORATORY EXERCISE #9 Rev: 3/10/10 History A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. [28] However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis [29] [30] . He was awarded the Nobel Prize in Chemistry in 1993 for his invention, [2] seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle. (see main article: Kary Mullis ) At the time he developed PCR in 1983, Mullis was working in Emeryville , California for Cetus Corporation , one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car [29] . 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 an enzyme called DNA polymerase . Mullis credits the psychedelic drug LSD for his invention of the technique. [1] ( Video ) In Scientific American , Mullis summarized the accomplishment: "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." [31] DNA polymerase occurs naturally in living organisms. In cells it functions to duplicate DNA when cells divide in mitosis and meiosis . Polymerase works by binding to a single DNA strand and creating the complementary strand. In the first of many original processes, the enzyme was used in vitro (in a controlled environment outside an organism). The double- stranded DNA was separated into two single strands by heating it to 94°C (201°F). At this temperature, however, the DNA polymerase used at the time were destroyed, so the enzyme had to be replenished after the heating stage of each cycle. The original procedure was very inefficient, since it required a great deal of time, large amounts of DNA polymerase, and continual attention throughout the process. In 1986, this original PCR process was greatly improved by the use of DNA polymerase taken from thermophilic bacteria grown in geysers at a temperature of over 110°C (230°F). The DNA polymerase taken from these organisms is stable at high temperatures and, when used in PCR, does not break down when the mixture was heated to separate the DNA strands. Since there was no longer a need to add new DNA polymerase for each 1
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cycle, the process of copying a given DNA strand could be simplified and automated. One of the first thermostable DNA polymerases was obtained from
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09x_PCR wikiped extras_120309 - POLYMERASECHAINREACTION

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