v=_YgXcJ4n-kQ - home.htm (PRC and qPCR real time PCR)
• 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.  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.
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.
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.
• 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.
Polymerase Chain Reaction PCR – Targets – Denaturing – Primers – Annealing – Cycles – Requirements
30–35 cycles each comprising: – denaturation (95°C), 30 sec. – annealing (55–60°C), 30 sec. – extension (72°C), time depends on product size
Thermal Cyclers •PCR cyclers available from many suppliers. •Many block formats and multi-block systems. •Reactions in tubes or 96-well micro-titre plates.
• 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)
Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C.
Primers range from 15 to 30 nucleotides, are single- stranded, and are used for the complementary building blocks of the target sequence.
- Fall '12
- DNA, Kary Mullis, PCR Primers