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Pfu DNA Polymerase as a Hot Start Alternative

which exhibits minimal polymerase activity at lower, nonstringent primer-annealing temperatures, is an effective solution for reducing the mispaired primer-extension reactions that occur in later PCR cycles.

Thermostability of Pfu DNA Polymerase: Amplifying GC-Rich Targets

In addition to exhibiting a relatively high Topt, Pfu DNA polymerase shows remarkable thermostability when compared to other thermophilic DNA polymerases.3 For example, Pfu DNA polymerase has a half-life of 18 to 25 hours at 95oC, while Taq DNA polymerase has a half-life of less than 1 hour (data not shown). The extreme thermostability of Pfu DNA polymerase allows higher denaturation temperatures to be used, which may increase the yield and purity of the desired PCR product. Specific research examples include the successful amplification of GC-rich PCR targets in the following human genes: dopamine D4 receptor gene (D4DR),10 proopiomelanocortin gene (POMC),10 and fragile X site in Xq27.3.11 These successful PCR amplifications were accomplished using Pfu DNA polymerase and a denaturation temperature of 98oC.

Conclusions

The following properties of Pfu DNA polymerase make it ideally suited for many PCR applications: (1) the lowest error rate of any thermostable DNA polymerase analyzed to date,1-2 which is an advantage in studies that require cloning, sequencing, gene expression and site-directed mutagenesis; (2) can be used alone to amplify relatively long PCR targets (up to 12 to 25 kb) if given adequate extension time,12 which is important if long gene sequences or vector constructs must be correctly amplified for further sequence analysis; (3) unusually high thermostability allows Pfu DNA polymerase to successfully amplify GC-rich PCR targets without the addition of adjuncts (such as D
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Related biology technology :

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4. Unique DNA Polymerase Formulation Excels in a Broad Range of PCR Applications
5. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
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9. The Use of MasterTaq Polymerase for PCR with Humic Material-Contaminated DNA
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11. C. therm. Polymerase One-Step RT-PCR System
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