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Enzymes for Molecular Biology

PCR and DNA Amplification

Taqs like it hot and some like it hot from the start

Most in vitro amplification applications use a thermostable enzyme for the polymerase chain reaction (PCR). The most used PCR enzyme is Taq DNA polymerase. Taq DNA polymerase is derived from Thermus aquaticus, an extremely thermophilic eubacterium which grows at temperatures above 70°C. This means that the optimal enzymatic activity of Taq DNA polymerase is expressed at approximately 72°C. 

Standard PCR or hot-start?

Standard Taq polymerase expresses optimal activity at 72°C but the enzyme becomes active from around 37°C. The low-temperature activity is substantial and has the disadvantage of permitting nonspecific amplification and mis-priming events during the initial lower-temperature phase of a PCR reaction. If your needs include automation of PCR, then consider using a hot-start Taq polymerase.

Hot-start polymerases, unlike standard Taq, are unreactive at ambient temperatures. Hot-start PCR techniques follow the same principles as conventional PCR, but enzymatic activity is suppressed until the first denaturation step has been accomplished. This minimizes nonspecific binding and mis-priming and results in improved specificity. In addition, the technology offers the significant convenience of reaction set up at room temperature making PCR automation possible. In some cases, even sensitivity is improved, because non-specific amplification depletes reaction components required for specific target amplification.

All Taq polymerases are suitable for:

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Enzymes for PCR applications

Choose a Taq DNA polymerase based on your requirements for standard PCR or for a hot-start enzyme suitable for automation and high throughput PCR applications.
* Stoffel Fragment Taq DNA polymerase is recommended for genotyping and primer extension. The Stoffel deletion (5’→3’ exo-) makes the enzyme slightly more thermostable with slightly greater fidelity than full length Taq and is strongly suggested for GC rich and secondary structure templates. In quantitative real-time PCR assays, TaqMan-type probes must be hydrolyzed during PCR by the 5'-nuclease activity of Taq DNA polymerase to generate a signal. The hybridization probes in Light-Cycler assays must not be hydrolyzed therefore accuracy of quantitative measurements is improved using the Stoffel Fragment that lacks 5'-nuclease activity.

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FAQs about enzymes for PCR and DNA amplification

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