KAPAHiFi™ HotStart DNA Polymerase is a novel, single-enzyme system that exhibits industry-leading performance when compared with other high fidelity polymerases and polymerase blends. The HotStart formulation increases reaction efficiency and sensitivity by eliminating spurious amplification products resulting from non-specific priming events during reaction setup and initiation.

Processivity is defined as the number of nucleotides incorporated by the polymerase per binding event, and is a major determinant of extension speed and robustness. KAPAHiFi™ HotStart has been engineered to have an increased affinity for DNA without the need for accessory protein domains. The intrinsic high processivity of KAPAHiFi™ HotStart results in significant improvements to yield, sensitivity, speed, target length, and the ability to amplify difficult templates.

Typical proofreading polymerases require extension times of 1 - 2 minutes per kilobase. KAPAHiFi™ HotStart requires extension times of just 30 seconds per kilobase, providing the potential to save up to 75% of total reaction time.

KAPAHiFi™ HotStart also possesses a strong 3’-5’ exonuclease-dependent proofreading activity resulting in superior accuracy.

KAPAHiFi™ HotStart kits include two buffers for optimal performance with difficult templates. Both buffers contain magnesium chloride allowing for convenient setup and robustness.

Product Applications:

KAPAHiFi™ HotStart is designed for high fidelity PCR where amplified product is cloned for use in downstream applications such as:

Site-directed mutagenesis
Sequencing
Protein expression

Amplicons generated with KAPAHiFi™ HotStart are suitable for routine downstream applications, including restriction enzyme digestion, blunt-end cloning, and sequencing.

Extreme fidelity:

The increased processivity, strong proofreading activity, and optimized buffer system of the engineered KAPAHiFi™ HotStart DNA Polymerase results in superior accuracy for high fidelity PCR applications.

Figure 1. Using a modified lacI-based fidelity assay, KAPAHiFi™ HotStart DNA Polymerase displays an error rate at least 30x lower than wild-type Taq, 10x lower than common high fidelity polymerase blends, and 3x lower than wild-type Pfu.

Highest yield and sensitivity from long genomic target:

Two-enzyme blend systems commonly used for long range applications are not suitable for high fidelity PCR because the error rates of polymerase blends are only 2 - 3x better than that of wild-type Taq (Figure 1).

The extreme processivity and robustness of KAPAHiFi™ HotStart DNA Polymerase offers the ability to perform high fidelity PCR on long and complex genomic templates with extreme sensitivity. The high speed of KAPAHiFi™ HotStart also allows significantly shorter reaction times for long range PCR.

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Figure 2. Amplification of hgDNA targets up to 11 kb. Each target was amplified from a descending range of template hgDNA concentrations (50 ng to 0.5 ng per reaction). Reactions (25 µl each) were performed using standard 3-step cycling profiles (35 cycles): 20 sec denaturation, 15 sec annealing, and 30 sec/kb extension time. Total reaction time for the 11 kb amplicon was 3h 50mins. 12.5 µl of each reaction was loaded on the gel.

Unrivalled success with difficult templates:

KAPAHiFi™ HotStart DNA Polymerase exhibits robust performance on difficult templates. A panel of amplicons from human genomic DNA with a GC content ranging from 47% to 84% was used to compare the performance of KAPAHiFi™ HotStart and competitor kits. A polymerase with fusion technology in GC Buffer and a polymerase blend are only able to amplify targets up to 64% GC content. KAPAHiFi™ HotStart in GC Buffer achieves a 100% success rate with higher yields across all amplicons up to 84% GC content.

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Figure 3. Amplification of AT- and GC-rich, single-copy human genomic targets. Seven single-copy gene fragments representing a range of GC content were used to compare the robustness of KAPAHiFi™ HotStart DNA Polymerase against a panel of competitor high fidelity DNA polymerases and polymerase blends. Amplicons range between 0.5 kb and 0.7 kb in length and ranged from 47% to 84% GC content. All reactions (25 µl each) contained 50 ng human genomic DNA as template and were performed using manufacturers’ protocols and buffers, with standard 3-step cycling profiles (35 cycles). 12.5 µl of each reaction was loaded on the gel.

Extreme sensitivity and fidelity:

A major limitation for single-enzyme proofreading polymerases is poor sensitivity due to damaged nucleotides and primer degradation. The engineered KAPAHiFi™ HotStart DNA Polymerase exhibits dramatic improvements in sensitivity - outperforming fusion technology polymerases and polymerase blends.

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Figure 4. 2 kb lambda phage target amplified from a 10 fold dilution series starting from 10 ng down to 10 fg using KAPAHiFi™ HotStart DNA Polymerase with Fidelity Buffer, fusion technology polymerase, and polymerase blend systems. All reactions (25 µl each) were performed using manufacturers’ protocols and buffers, with standard 3-step cycling profiles (35 cycles). 12.5 µl of each reaction was loaded on the gel.