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prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

ase fidelity contributes significantly to the accuracy of RT-PCR, we measured the frequency of mutations produced when a cDNA synthesis reaction mixture was amplified using different PCR enzymes. We modified our PCR-based forward mutation assay7 such that the lacI target gene (1.9-kb lacIOlacZa fragment) was amplified from cDNA synthesized from in vitro transcribed mRNA, rather than from plasmid DNA. RT-PCR products containing lacI were cloned into lambda arms, and the percentage of lacI mutants was determined in a color-screening assay.7,10 Mutation frequencies were lowest in amplifications carried out with Pfu DNA polymerase (Table 1). In contrast, Taq DNA polymerase and the TaqPlus Long DNA polymerase blend produced mutation frequencies that were 5- and 3-fold higher, respectively. Therefore, DNA polymerase fidelity contributes to the accuracy of RNA amplification and should be an important consideration when choosing a PCR enzyme for RT-PCR procedures, particularly when the product is to be cloned, sequenced, and/or expressed.

Stratagenes Systems Include High-Fidelity PCR Enzymes

The ProSTAR RT-PCR systems features Stratagenes high performance/high fidelity PCR enzymes, PfuTurbo DNA polymerase and TaqPlus Precision DNA polymerase blend. PfuTurbo DNA polymerase, a blend of cloned Pfu DNA polymerase and a novel thermostable PCR enhancing factor, amplifies PCR products in higher yield than Taq DNA polymerase,8 and other proofreading single-enzyme formulations.9 Moreover, Pfu and PfuTurbo DNA polymerases exhibit significantly greater fidelity than all other PCR enzymes and DNA polymerase blends.7,9 Stratagenes TaqPlus Precision PCR system is a Taq and Pfu DNA
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