High-fidelity enzymes generally utilize a 3'→5' exonuclease activity to remove incorrectly incorporated bases. However, this activity can also degrade primers during setup and the start of PCR, which can result in nonspecific priming, smearing, or even failure to amplify any product at all — especially when using low amounts of template.
HotStar HiFidelity DNA Polymerase
Uniquely isolated from a novel Pyrococcus spp. source, HotStar HighFidelity DNA Polymerase is chemically modified to inactivate both the polymerase and the exonuclease activity. A simple 5-minute hot start activates both enzymatic components, allowing PCR to start with high proofreading ability. This unique modification ensures that primers and template remain intact during setup, preventing mispriming. The reliable performance of HotStar HiFidelity DNA Polymerase ensures highly specific and sensitive amplification.
HotStar HiFidelity PCR Buffer
Novel HotStar HiFidelity PCR Buffer contains the proprietary PCR additive, Factor SB, as well as optimized concentrations of dNTPs and MgSO4. Factor SB is a unique PCR additive that improves PCR sensitivity and reliability (see figure "Highly sensitive and reliable PCR"). Innovative Factor SB ensures that the template DNA, which is often degraded by the 3'→5' exonuclease activity of high-fidelity enzymes, remains intact.
The preoptimized formulation provides reliable amplification of specific PCR products with a very low error rate. The buffer accomplishes this by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, HotStar HiFidelity PCR Buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.
HotStar HiFidelity PCR Buffer also contains specifically optimized concentrations of dNTPs for convenient PCR setup and reliable results. The buffer also maximizes the fidelity of HotStar HiFidelity DNA Polymerase, which ensures a very low error rate. The HotStar HiFidelity Polymerase Kit is highly suitable for any high-fidelity PCR application due to the increased sensitivity, specificity, and minimal optimization requirements afforded by the HotStar HiFidelity PCR Buffer.
The HotStar HiFidelity Polymerase Kit includes Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates (see figure "Amplification of difficult templates"). Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. Adding Q-Solution to the PCR does not compromise PCR fidelity.
Complete kit format
The HotStar HiFidelity Polymerase Kit includes everything required for PCR. No additional reagents or buffers need to be purchased separately.