The Polymerase Chain Reaction Biotechnology

Polymerase Chain Reaction Amplification of DNA in vitro Developed in 1984 by Kary Mullis (who worked for Cetus Biotechnology) Cruizing the Pacific Coast Highway from San Francisco to Mendocino on a motorcycle $10,000 bonus 1991 patent was sold to LaRoche for 300 million Something like it was suggested but never tested by Gobind Khorana several years earlier described a method to replicate a region of DNA using DNA pol and 2 primers

PCR Components Template DNA that contains the target sequence or amplicon Primers Short 20-30 nucleotides with sequences complimentary to the amplicon Used in pairs, as the forward primer and the reverse primer they define the endpoints of the amplified region Usually designed such that Tm between 55 and 72C 3’ ideally with a high GC content No complimentary or palindromic sequences

PCR Components Continued dNTPs Nucleotides GATC added in equal concentrations DNA polymerase Heat-resistant Taq (Thermophilus aquaticus) polymerase Magnesium chloride Cofactor for DNA polymerase Needed for optimal activity Buffer Provides optimal conditions for the enzyme pH, salt concentration, etc.

Thermocycler: Reaction Cycle Denaturing step Denatures the double stranded DNA into single strands Annealing step Allows the primers to attach to complementary strand of DNA Extension step Optimal temperature for Taq polymerase to attach to and extend the new strand of DNA These steps are repeated for 20-30 cycles

PCR Summary PCR allows you to amplify DNA Can amplify specific gene Copies are made of sequence between the two primers New copies each serve as templates Amount of DNA doubles in each cycle - increases exponentially 2n where n = # of cycles 30 cycles = 230 = 1x 109 copies of DNA - Can amplify up to a billion-fold! Part of the nucleotide sequence must be known

Optimizing the PCR Reaction Annealing temperature of the primers. The concentration of Mg2+ in the reaction. The duration and temperature of each step The amount of template and polymerase “more is less” Fidelity of the Reaction Taq DNA polymerase lacks the 3´→5´ proof-reading activity commonly present in other polymerases. Taq mis-incorporates 1 base in 104. Error distribution will be random.

Optimizing the PCR Reaction How many cycles? Increasing the cycle number above ~35 has little positive effect. The plateau occurs when: The reagents are depleted The products re-anneal The polymerase is damaged Unwanted products accumulate.

Special Considerations Because a small amount of DNA can be amplified in a polymerase chain reaction, contamination can be a problem Use sterile techniques A negative control is very important (today’s lab we will use water as the template)