1. The Polymerase Chain Reaction
Biotechnology

2. 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

3. PCR Components Template
DNA that contains the target sequence or amplicon
Primers
Short 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

4. 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.

5. 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 cycles

7. 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

8. 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.

9. 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.

10. 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)