Polymerase Chain Reaction

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Information about Polymerase Chain Reaction

Published on September 26, 2008

Author: rkduary



Slide 1: Polymerase Chain Reaction Raj Kumar Duary Molecular biology unit, Dairy microbiology Division, National Dairy Research Institute Karnal, India-132001 E-mail : Specific synthesis of DNA in vitro via a PCR reaction. (Emeryville; California, Cetus) : Specific synthesis of DNA in vitro via a PCR reaction. (Emeryville; California, Cetus) Invented by- Kary Mullis (1983) Shared the Nobel Prize in Chemistry with Michael Smith in 1993. Slide 3: PCR is a rapid, inexpensive and simple way of copying specific DNA fragments from minute quantities of source DNA material • It does not necessarily require the use of radioisotopes or toxic chemicals • It involves preparing the sample DNA and a master mix with primers, followed by detecting reaction products Slide 4: Chain reaction relies on DNA replication process Repeated cycles of melting (strand separation), primer annealing, and primer extension by cycling temperatures Need a tough enzyme to deal with high temperatures Polymerases isolated from thermophilic bacteria (Thermus aquaticus, Pyrococcus furiosus) Slide 5: ! Denaturation: DNA fragments are heated at high temperatures, which reduce the DNA double helix to single strands. These strands become accessible to primers ! Annealing: The reaction mixture is cooled down. Primers anneal to the complementary regions in the DNA template strands, and double strands are formed again between primers and complementary sequences ! Extension: The DNA polymerase synthesizes a complementary strand. The enzyme reads the opposing strand sequence and extends the primers by adding nucleotides in the order in which they can pair. The whole process is repeated over and over PCR procedures: steps Slide 6: Melting temperature Tm°C = 2(A/T) + 4(G/C) Tm oC Temperature at which half possible H bonds are formed Slide 7: The Thermus aquaticus DNA polymerase (Taq) Not permanently destroyed at 94ºC Optimal temperature is 72ºC Does not have proof reading ability Error rate 1 in 2 X 104 bases Seems rare but can be recovered in cloning a single molecule Newer polymerases have high fidelity : Does not have proof reading ability Error rate 1 in 2 X 104 bases Seems rare but can be recovered in cloning a single molecule Newer polymerases have high fidelity Problems with Taq Slide 9: 

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