vYgXcJ4n kQ homehtm PRC and qPCR real time PCR

Vygxcj4n kq homehtm prc and qpcr real time pcr

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v=_YgXcJ4n-kQ - home.htm (PRC and qPCR real time PCR)
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The Invention of PCR • Invented by Kary Mullis in 1983. • First publishedaccount appeared in 1985. • Awarded Nobel Prize for Chemistry in 1993. Michael Smith, Kary banks Mullis In 1983, Mullis was working for Cetus Corp.as a chemist.That spring, according to Mullis, he was driving his vehicle late one night with his girlfriend, who was also a chemist at Cetus, when he had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times. Cetus took Mullis off his usual projects to concentrate on PCR full-time,and Mullis spent more than a year trying to show his idea would work, but could not produce "definitive proof" of the concept. Mullis eventually succeeded on December 16, 1983. [3] In his Nobel Prize lecture, he remarked that the success didn't make up for his girlfriend breaking up with him shortly before: "I was sagging as I walked out to my little silver Honda Civic. Neither [assistant] Fred, empty Beck's bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome. He received a $10,000 bonus from Cetus for the invention.
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Did He Really Invent PCR? • The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: – Kleppe et al. (1971) J. Mol. Biol. 56, 341-346. • Progress was limited by primer synthesis and polymerase purification issues. • Mullis properly exploited amplification.
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PCR- Polymerase Chain Reaction PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing. What is the Polymerase Chain Reaction? • It’s a means of selectively amplifying a particular segment of DNA. • The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene. • It can be thought of as a molecular photocopier.
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How Powerful is PCR? • PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. • The template DNA need not be highly purified — a boiled bacterial colony. • The PCR product can be digested with restriction enzymes, sequenced or cloned. • PCR can amplify a single DNA molecule, e.g. from a single sperm.
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Polymerase Chain Reaction PCR Targets Denaturing Primers Annealing Cycles Requirements
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30–35 cycles each comprising: – denaturation (95°C), 30 sec. – annealing (55–60°C), 30 sec. – extension (72°C), time depends on product size
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Thermal Cyclers •PCR cyclers available from many suppliers. •Many block formats and multi-block systems. •Reactions in tubes or 96-well micro-titre plates.
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What’s in the Reaction? • Template DNA • Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium ions,bovine serum albumin) • Nucleotides (dNTPs) • Primers • DNA polymerase (usually Taq)
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Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C.
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Primers range from 15 to 30 nucleotides, are single- stranded, and are used for the complementary building blocks of the target sequence.
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  • DNA, Kary Mullis, PCR Primers

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