Maximizing PCR and RT-PCR Success - Enzymes

PCR Brochure 10/2016 3 Taq DNA polymerase Several types of thermostable DNA polymerases are available for use in PCR, providing a choice of enzymatic properties (Table 1). Taq DNA polymerase, isolated from the eubacterium Thermus aquaticus, is the most commonly used enzyme for standard end-point PCR. The robustness of this enzyme allows its use in many different PCR assays. However, as this enzyme is active at room temperature, it is necessary to perform reaction setup on ice to avoid nonspecific amplification. QIAGEN has overcome this limitation with the introduction of the novel TopTaq® DNA Polymerase. This innovative non-hot-start enzyme has limited access to primer and template at room temperature, allowing immediate reaction setup without the use of ice. A number of modifications of the original “PCR polymerase” – Taq DNA polymerase – are now available for different downstream application needs, such as hot-start, single-cell, or multiplex PCR (see page 16). With an average error rate of 1 in 10,000 nucleotides, Taq DNA polymerase and its variants are less accurate than the thermostable enzymes of DNA polymerase family B. However, due to its versatility, Taq DNA polymerase is still the enzyme of choice for most routine applications and when used with a stringent hot- start, is suitable for several challenging PCR applications (Table 4, page 16). Hot-start PCR polymerase When amplification reaction setup is performed at room temperature, primers can bind nonspecifically to each other, forming primer–dimers. During amplification cycles, primer– dimers can be extended to produce nonspecific products, which reduces specific product yield. For more challenging PCR applications, the use of hot-start PCR is crucial for successful specific results. To produce hot-start DNA polymerases,TaqDNApolymeraseactivitycanbeinhibitedat lower temperatures with antibodies or, even more effectively, with chemical modifiers that form covalent bonds with amino acids in the polymerase. The chemical modification leads to complete inactivation of the polymerase until the covalent bonds are broken during the initial heat activation step. The unique hot-start procedure, based on chemical modification, provided with QIAGEN® hot-start enzymes is easily incorporated into any PCR program using a simple 5-minute (HotStarTaq® Plus DNA Polymerase) or 15-minute (HotStarTaq DNA Polymerase) initial denaturation step (Figure 2, next page). Enzymes Table 1. DNA polymerases used in PCR* * Review article: Ishino, S. and Ishino, Y. (2014) DNA polymerases as useful reagents for biotechnology – the history of developmental research in the field. Front Microbiol. 5, 465. † For example, TopTaq DNA Polymerase. ‡ For example, HotStarTaq Plus DNA Polymerase. § HotStar HiFidelity DNA Polymerase provides A-addition action for easy TA/UA-cloning DNA polymerase family A DNA polymerase family B Enzymes available Taq DNA Polymerases,† Hot-start DNA polymerases‡ Proofreading enzymes§ 5'–3' exonuclease activity + – 3'–5' exonuclease activity – + Extension rate (nucleotides/second) ~ 150 ~ 25 Error rate (per bp/per cycle) 1 in 103 /104 1 in 105 /106 PCR applications Standard, hot-start, reverse transcription, real-time High fidelity, cloning, site-directed mutagenesis A-addition + Sometimes§

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