HotStar HiFidelity Polymerase Kit

HotStar HiFidelity Polymerase Kit includes a hot-start proofreading enzyme uniquely modified to prevent degradation of primers and template during PCR setup, providing reliable high-fidelity PCR without the need for optimization. The HotStar HiFidelity DNA Polymerase Kit outperformed kits tested from other suppliers and ensures highly specific and sensitive amplification (see figure "Highly sensitive and reliable PCR"). HotStar HiFidelity DNA Polymerase fidelity is over 10-times higher than Taq DNA polymerase. Proprietary Factor SB contained in the novel HotStar HiFidelity PCR Buffer improves the sensitivity and reliability of PCR, especially when low amounts of starting template are used. Suboptimal PCR can be improved with Q-Solution, also provided with the kit (see figure "Amplification of difficult templates").

HotStar HiFidelity DNA Polymerase specifications

Concentration: 2.5 units/μl 
5'–>3' exonuclease activity: No 
3'–>5' exonuclease activity: Yes
Extra A addition (terminal transferase activity): Yes
Half-life: >4 hours at 95°C
Nuclease contamination: No
Protease contamination: No
RNase contamination: No

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.  

Q-Solution

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.

The HotStar HiFidelity procedure is straightforward — simply follow the step-by-step, optimized protocol included with the kit. HotStar HiFidelity DNA Polymerase has been chemically modified to temporarily inactivate not only the polymerase activity, but also the 3'–>5' exonuclease activity of the enzyme. This prevents excessive degradation of primers and template during PCR setup and the initial PCR cycles. Both polymerase and proofreading activities are easily restored by a 5-minute, 95°C incubation step, which can be readily incorporated into the PCR cycling program. This modification allows convenient room-temperature reaction setup without compromising PCR specificity or product yield.

TA/UA cloning

In contrast to all other high-fidelity DNA polymerases, PCR products generated using HotStar HiFidelity DNA Polymerase can be used directly in TA- or UA-cloning procedures (see figure "Efficient TA/UA cloning").

Product length and sensitivity

Due to the high sensitivity of the HotStar HiFidelity Polymerase Kit, it is possible to amplify fragments with very low amounts of starting material (e.g., as little as 1 ng human genomic DNA). Product lengths of up to 5 kb and 10 kb have been achieved using genomic DNA and lambda DNA respectively, thus indicating the suitability of the HotStar HiFidelity Polymerase Kit for any application, including the amplification of long DNA fragments for cloning.

HotStar HiFidelity Polymerase Kit is highly suitable for all hot-start PCR applications that require high sensitivity combined with a low error rate, such as cloning and site-directed mutagenesis.