KAPAHiFi DNA Polymerase is a novel, single-enzyme system that exhibits industry-leading performance when compared with other high fidelity polymerases and polymerase blends. Processivity is defined as the number of nucleotides incorporated by the polymerase per binding event, and is a major determinant of extension speed. In contrast to fusion technologies that attach DNA binding proteins to Pfu-like polymerases to improve processivity, KAPAHiFi DNA Polymerase has been engineered to have an increased affinity for DNA without the need for accessory protein domains. The intrinsic high processivity of KAPAHiFi results in significant improvements in yield, speed, target length, and the ability to amplify difficult templates.

Typical proofreading polymerases require extension times of 1 - 2 minutes per kilobase. KAPAHiFi DNA Polymerase requires extension times of just 30 seconds per kilobase, providing the potential to save up to 75% of total reaction time. KAPAHiFi also possesses a strong 3’-5’ exonuclease-dependent proofreading activity resulting in superior accuracy.

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

Product Applications

KAPAHiFi DNA Polymerase 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 the KAPAHiFi DNA Polymerase are suitable for routine downstream applications, including restriction enzyme digestion, blunt-end cloning, and sequencing.

Superior fidelity

Figure 1. Using a lacI-based fidelity assay, KAPAHiFi DNA Polymerase displays an error rate at least 15x lower than wild-type Taq, 5x lower than common polymerase blends (for long range PCR), and 2x lower than KOD/Pfx.

High yield and specificity from genomic DNA

Low yield and poor specificity are common challenges when amplifying from genomic DNA. KAPAHiFi DNA Polymerase in High Fidelity Buffer achieves high yield and specificity on human genomic DNA.

Figure 2. Amplification of a 2.7 kb single-copy target from human genomic DNA, using a descending range of template DNA concentrations (100 ng to 4 ng per reaction). Reactions (25 µl each) were assembled and performed according to manufacturers’ protocols, using standard 3-step cycling profiles (30 cycles). 20 µl of each reaction was loaded on the gel.

Unrivalled success with difficult templates

KAPAHiFi DNA Polymerase consistently exhibits robust performance on difficult templates. A panel of amplicons from human genomic DNA with a GC content ranging from 28% to 84% was used to compare the performance of KAPAHiFi and competitor kits. A polymerase with fusion technology in GC Buffer is only able to amplify targets up to 64% GC content. KAPAHiFi DNA Polymerase in GC Buffer achieves a 100% success rate with higher yields across all amplicons up to 84% GC content.

Figure 3. Amplification of AT- and GC-rich, single-copy human genomic targets. Eight single-copy gene fragments representing a range of GC content were used to compare the robustness of KAPAHiFi DNA Polymerase against a panel of competitor high fidelity DNA polymerases. Amplicons range between 0.4 kb and 0.7 kb in length and ranged from 28% to 84% GC content. All reactions (25 µl each) contained 100 ng human genomic DNA as template and were performed using manufacturers’ protocols and buffers, with standard 3-step cycling profiles (30 cycles). 5 µl of each reaction was loaded on the gel.

Long range and high fidelity

Two-enzyme blend systems commonly used for long range applications are often not suitable for high fidelity PCR because the error rate of blends is only 2 - 4x better than that of wild-type Taq (see Figure 1). The extreme processivity and robustness of KAPAHiFi DNA Polymerase offers the ability to perform high fidelity PCR on long templates. The high speed of KAPAHiFi allows significantly shorter reaction times for long range PCR.

Figure 4. Amplification of lambda phage targets up to 18 kb. Targets between 2 kb and 18 kb were amplified from 50 ng of lambda DNA in 25 µl reactions, using KAPAHiFi DNA Polymerase with High Fidelity Buffer and standard reaction conditions. Thermal cycling was performed using a standard 2-step protocol (30 cycles) and extension time of 30 sec/kb. 5 µl of each reaction was loaded on the gel.

KAPAHiFi DNA Polymerase Kit Components

KAPAHiFi DNA Polymerase (1 U/µl)
5x KAPAHiFi High Fidelity Buffer with MgCl₂
5x KAPAHiFi GC Buffer with MgCl₂
25 mM MgCl₂
KAPA dNTP Mix (10 mM each nucleotide)