1. Topics to be covered Basics of PCR
Biochemical activities of polymerases
Differences between polymerases, e.g. Taq versus Phusion
Applications for Polymerases
Mutagenesis, error-prone PCR, overlap-PCR

2. Polymerase Chain Reaction (PCR)
PCR is essentially DNA replication in vitro
The PCR can copy segments of DNA by up to a billionfold in the test tube during 1-2 hours
This process called amplification
Such large amounts of specific genes or other DNA segments may be used in many molecular biology applications

3. PCR
PCR uses the heat-stable enzyme DNA polymerase, which naturally copies DNA molecules
Artificially synthesized DNA primers are used to initiate DNA synthesis
PCR does not actually copy whole DNA molecules but amplifies stretches of up to a few thousand base pairs (the target) from within a larger DNA molecule (the template)
PCR was invented in 1980s by Kary Mullis, who got a Nobel Prize for this achievement

4. PCR is a powerful tool
It is easy to perform, extremely sensitive, specific, and highly efficient
During each round of amplification the amount of product doubles, leading to an exponential increase in the DNA
This means not only that a large amount of amplified DNA can be produced in just a few hours, but that only a few molecules of target DNA need be present in the sample to start the reaction

5. Polymerase chain reaction
PCR is a biochemical technology used in molecular biology to amplify a specific DNA fragment from a single or a few copies of a piece of DNA
PCR applications employ a heat- stable DNA polymerase, with or w/o proofreading activity
PCR uses thermal cycling
Melting of template DNA -> single strand
Annealing of oligonucleotides
Extension of 3’ ends by polymerase
Increase genetic variability by
using polyermases lacking proofreading activity
Using conditions, which favor mutations (unbalance mix of dNTPs, Mg2+ concentration)
Melting at 95ºC
Annealing at 60ºC
Extension at 72ºC

6. PCR amplification can introduce sequence errors

7. Properties of commonly used thermostable DNA polymerases

8. PCR applications Obtaining DNA for cloning genes
DNA for sequencing purposes because the gene or genes of interest can easily be amplified if flanking sequences are known
Phylogenetic studies to amplify 16S rRNA genes from various environmental sources
Amplify and clone DNA from ancient and fossilized plants, animals etc
Diagnostic microbiology
Forensics - to identify human individuals from very small samples of their DNA

9. Generation of deletion fusions by overlapping PCR

10. Directed PCR mutagenesis

11. See also: https://www.neb.com/