2 Polymerase Chain Reaction Amplification of DNA in vitroDeveloped 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 bonus1991 patent was sold to LaRoche for 300 millionSomething like it was suggested but never tested by Gobind Khorana several years earlierdescribed 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 ampliconPrimersShort nucleotides with sequences complimentary to the ampliconUsed in pairs, as the forward primer and the reverse primer they define the endpoints of the amplified regionUsually designed such thatTm between 55 and 72C3’ ideally with a high GC contentNo complimentary or palindromic sequences
4 PCR Components Continued dNTPsNucleotides GATC added in equal concentrationsDNA polymeraseHeat-resistant Taq (Thermophilus aquaticus) polymeraseMagnesium chlorideCofactor for DNA polymeraseNeeded for optimal activityBufferProvides optimal conditions for the enzymepH, salt concentration, etc.
5 Thermocycler: Reaction Cycle Denaturing stepDenatures the double stranded DNA into single strandsAnnealing stepAllows the primers to attach to complementary strand of DNAExtension stepOptimal temperature for Taq polymerase to attach to and extend the new strand of DNAThese 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 primersNew copies each serve as templatesAmount of DNA doubles in each cycle - increases exponentially2n where n = # of cycles30 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 stepThe amount of template and polymerase “more is less”Fidelity of the ReactionTaq 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 depletedThe products re-annealThe polymerase is damagedUnwanted products accumulate.
10 Special Considerations Because a small amount of DNA can be amplified in a polymerase chain reaction, contamination can be a problemUse sterile techniquesA negative control is very important (today’s lab we will use water as the template)