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

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

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

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

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

PCR amplification can introduce sequence errors

Properties of commonly used thermostable DNA polymerases

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

Generation of deletion fusions by overlapping PCR

Directed PCR mutagenesis

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