Polymerase Chain Reaction. Invented by Kary Mullis Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.

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Polymerase Chain Reaction

Invented by Kary Mullis Mullis and Faloona, Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Nobel Prize 1993

“I was working for Cetus, making oligonucleotides. They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. I was driving with Jennifer Barnett to a cabin I had been building in northern California. She and I had worked and lived together for two years. She was an inspiration to me during that time as only a woman with brains, in the bloom of her womanhood, can be. That morning she had no idea what had just happened. I had an inkling. It was the first day of the rest of my life.” - from Karry Mullis’s autobiography at the Nobel e- Museum

Specifically targets and amplifies a SINGLE sequence from within a complex mixture of DNA. How is this different from cloning?

Takes advantage of basic requirements of replication A DNA template Nucleotides Primers polymerase PCR is DNA replication in a test tube

Primers Must have some information about sequence flanking your target Primers provide specificity

Complementary to opposite strands with 3’ ends pointing towards each other Should have similar melting temperatures Be in vast excess

Melting temperature Tm oC = 2(A/T) + 4(G/C) Tm oC Temperature at which half possible H bonds are formed

Steps of PCR

Annealing then primer elongation

Thermocycling 94 degrees 55 degrees 70 degree

Heat-stable polymerase is vital to the ease of the process…

Problems with Taq

Automated long time ago…

Problems with PCR Contamination Takes one mismatch early on to amplify the wrong fragment