Case Study: Cancer Resequencing on Roche GS FLX

KAPA HiFi DNA Polymerase is confirmed as the world's highest fidelity polymerase for PCR. Lowest error rate and highest yield decrease false positives and increase coverage for cancer SNP detection. Download here >>

High-fidelity GC-rich PCR

The increased processivity of the KAPA HiFi DNA Polymerase allows for high-fidelity amplification of difficult templates with GC content up to 85%. Download the new application note on High-fidelity GC-rich PCR. Download here>>

Site-directed Mutagenesis

For more information on using KAPA HiFi for site-directed mutagenesis download the application note. Download here>>

Product Description

KAPA HiFi DNA Polymerase is a novel proofreading polymerase that was the engineered to have high processivity, exhibiting significant improvements in yield, sensitivity, speed, target length, and ability to amplify difficult templates (e.g GC-rich) when compared to wild-type polymerases. The HotStart formulation of the enzyme increases reaction efficiency and sensitivity by eliminating spurious amplification products resulting from non-specific priming events during reaction setup and initiation.

KAPA HiFi PCR Kits include two buffers for optimal performance with difficult templates. Both buffers contain magnesium chloride allowing for convenient setup and robustness.

Product Applications

KAPA HiFi 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
• Next-generation DNA resequencing

Amplicons generated with KAPA HiFi DNA Polymerase 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 KAPA HiFi DNA Polymerase results in superior accuracy for high fidelity PCR applications.

Figure 1. The error rate of KAPA HiFi is 100x lower than Taq polymerase, 40x lower than polymerase blends such as Platinum® Taq High Fidelity, 3x lower than Pfu Ultra, and 2x lower than Phusion.

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

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

KAPA HiFi 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. KAPA HiFi 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. 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 KAPA HiFi DNA Polymerase exhibits dramatic improvements in sensitivity - outperforming fusion technology polymerases and polymerase blends.

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.

Frequently Asked Questions

KAPA HiFi DNA Polymerase is an engineered B-family (proofreading) enzyme. KAPA HiFi enzyme and reaction buffers have very different characteristics to those of wild-type B-family DNA polymerases (e.g. Pfu, KOD and Vent) and buffers, and should not be evaluated using protocols recommended for other proofreading enzymes. For optimal results, it is important to follow the recommendations provided in the KAPA HiFi or KAPA HiFi HotStart Technical Data Sheet.

1. What are the most common causes of poor results (non-specific amplification, smearing or reaction failure) with KAPA HiFi?

• Inadequate denaturation. KAPA HiFi buffers have a significantly higher salt concentration that most other PCR buffers. To ensure adequate template denaturation, always denature for 20 sec at 98 °C in each PCR cycle.
• Suboptimal annealing temperature. Due to the high salt concentration of KAPA HiFi Buffers, optimal annealing temperatures are typically higher that they would be for the same primers using a different enzyme system. Annealing temperatures <60 °C are not recommended, as they lead to non-specific amplification. Start with an annealing temperature of 65 °C when using KAPA HiFi Fidelity Buffer, or 60 °C when using KAPA HiFi GC Buffer. If non-specific products are obtained, determine the optimal annealing temperature in a Ta gradient PCR (60 – 78 °C). Because of high annealing temperatures, 2-step protocols with a combined annealing/extension step at 68 – 78 °C may be used.
• Too long extension times. Use 30 sec/kb extension time per cycle for amplicons between 1 and 10 kb. For amplicons ≤1 kb, use 15 – 30 sec per cycle. For amplicons >10 kb, use 30 – 60 sec/kb per cycle.
• Suboptimal amount of template. Too much template is likely to lead to non-specific amplification. Use ≤25 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µl reaction.
• Too much enzyme. Use 0.5 U enzyme per 25 µl reaction.
• Suboptimal primer concentration. Use 0.3 mM of each primer. Lower primer concentrations are likely to result in low yields or smearing. Higher primer concentrations will increase primer-dimer formation and non-specific amplification.
• Suboptimal dNTP concentration. Use a final concentration of 0.3 mM of each dNTP (1.2 mM total dNTPs).
• Suboptimal dNTP quality. B-family (proofreading) DNA polymerases are more sensitive than A-family DNA polymerases (e.g. Taq) to dUTP poisoning. Only use high quality dNTPs supplied by KAPA Biosystems with KAPA HiFi enzymes.

 2. What are the standard cycling conditions for KAPA HiFi and KAPA HiFi HotStart?
3-step cycling profile (for optimal Ta in the range of 60 – 70 °C):

• Initial denaturation: 2 – 5 min at 95 °C
• Denaturation: 20 sec at 98 °C
• Annealing: 15 sec at optimal Ta (60 – 70 °C)
• Extension: 15 – 30 sec/kb at 72 °C
• Final extension: 1 – 5 min at 72 °C
• Number of cycles: 15 – 35

2-step cycling profile (for optimal Ta in the range of 68 – 78 °C):

• Initial denaturation: 2 – 5 min at 95 °C
• Denaturation: 20 sec at 98 °C
• Annealing/extension: 20 – 45 sec/kb at 68 – 78 °C
• Final extension: 1 – 5 min at 72 °C
• Number of cycles: 15 – 35

 
3. For which applications should I use KAPA HiFi PCR Kits?

• High-fidelity amplification of DNA to be cloned for sequencing, mutation analysis or protein expression.
• Site-directed mutagenesis.
• High performance resequencing sample enrichment for next-generation sequencing applications.
• High-fidelity amplification of GC-rich and other difficult templates.

4. Why is KAPA HiFi DNA Polymerase the preferred enzyme for high-fidelity PCR?
KAPA HiFi DNA Polymerase is a novel, single-enzyme system that has been engineered for high fidelity and increased processivity. Processivity is defined as the number of nucleotides incorporated by a DNA polymerase per binding event and is a major determinant of extension speed, robustness and sensitivity. KAPA HiFi has an increased affinity for DNA without the need for accessory protein domains. The intrinsic high processivity results in significant improvements to yield, sensitivity, speed, target length, and the ability to amplify difficult templates. Furthermore, KAPA HiFi DNA Polymerase possesses strong 3'-5' exonuclease (or proofreading) activity and has the lowest published error rate (i.e. highest fidelity) of all commercially available B-family DNA polymerases.

 5. Does it matter whether I order KAPA HiFi or KAPA HiFi HotStart?
KAPA HiFi HotStart is comprised of KAPA HiFi DNA Polymerase and a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency and sensitivity. Both enzymes are supplied with the same two reaction buffers, and have the same fidelity when used in KAPA HiFi Fidelity Buffer.

6. Which KAPA HiFi buffer should I use for my assay?
KAPA HiFi and KAPA HiFi HotStart are supplied with two 5X reaction buffers, namely KAPA HiFi Fidelity Buffer and KAPA HiFi GC Buffer.

KAPA HiFi Fidelity Buffer is recommended as the first approach for most applications. The GC Buffer should be used:

• for targets with a high GC content (>65%) or stable secondary structure
• to improve the yield and/or specificity of the target amplicon
• Both buffers contain Mg at a 1X concentration of 2.0 mM. Please note the following important differences between the two buffers:
• The Fidelity Buffer will produce a 2-fold lower error rate than the GC Buffer in the same assay. The Fidelity Buffer is therefore recommended for all applications where the highest fidelity is required, except for amplicons that fail to amplify in this buffer.
• Optimal annealing temperatures are likely to be lower in the GC Buffer than the Fidelity Buffer due to the presence of DNA destabilizers in the GC Buffer.

7. Can I still use KAPA HiFi DNA Polymerase if my existing assay requires a specialized buffer?
KAPA HiFi Buffers are unique and have been designed specifically for KAPA HiFi DNA Polymerase. KAPA HiFi and KAPA HiFi HotStart cannot be used in buffers designed for use with other B-family DNA polymerases. Likewise, KAPA HiFi Buffers cannot be used in combination with other PCR enzymes. Due to the unique properties and composition of KAPA HiFi enzymes and buffers, it is important to follow the recommendations for reaction setup and cycling parameters in the Technical Data Sheet to ensure optimal performance.

8. How can I ensure the highest possible fidelity (lowest error rate) in a PCR?
The error rate achieved in a PCR assay is dependent on characteristics of the DNA Polymerase, the chemical environment (e.g. buffer composition and Mg concentration), DNA template quality and number of PCR cycles. To ensure the highest fidelity in a particular assay:

• Use as much template as possible. 10 ng of plasmid/lambda DNA or 100 ng of genomic DNA is a good starting point. Please note that larger amounts of template may require extra Mg in the PCR reaction. Non-specific amplification associated with high template concentration may be controlled by reducing the number of cycles.
• Use as few PCR cycles as possible to obtain an adequate yield of the target amplicon for downstream applications. 25 cycles is a good starting point. Excessive cycling will lead to an accumulation of errors and may induce mutation through DNA damage.
• Use high quality template. Always dilute and store DNA templates in a buffered solution, e.g. 10 mM Tris pH 8.0 – 8.5 (with or without 1 mM EDTA).
• Minimize or eliminate the exposure of the PCR product to UV light.

9. What should I do if PCR products generated with KAPA HiFi enzymes contain a high background of non-specific amplicons or high molecular weight smears?
The increased processivity of KAPA HiFi DNA Polymerase may sometimes result in a higher background of non-specific amplicons or high-molecular weight smears when compared to products obtained with wild-type Taq or wild-type proofreading enzymes under similar conditions. Non-specific amplification and/or smearing can usually be eliminated by one, or a combination, of the following actions:

• Increase the annealing temperature. It is recommended that an annealing temperature gradient PCR be performed to determine the optimal Ta; especially in the case of primer sets that have a propensity to produce smearing and/or non-specific products. Certain primer sets might require a combined annealing/extension temperature in the range of 75 - 78 °C, especially where a complex genomic template is used.
• Decrease the annealing and/or extension time. An annealing time of 15 sec per cycle and an extension time of 30 sec/kb per cycle are recommended as a first approach. If smearing or non-specific amplification proves to be a problem, a reduction in the annealing time may improve results, particularly on slow-ramping (older) thermocyclers. If smearing or non-specific amplification products of a higher molecular weight than the target amplicon still occur, even after optimization of the annealing temperature and time, the extension time should be reduced to 15 sec/kb per cycle.
• Decrease the enzyme concentration. 0.5 U of KAPAHiFi HotStart enzyme per 25 µl reaction should be optimal for most assays. However, if smearing still occurs after the annealing temperature and annealing and extension times have been optimized, the amount of enzyme may be reduced in an attempt to obtain better results.
• Optimize the Mg concentration. Suboptimal Mg concentrations, especially excess Mg, may cause smearing and non-specific products. Typically, the 2 mM Mg included in KAPA HiFi Buffers at a 1X concentration is sufficient for most applications. Increasing the Mg concentration may improve results in some assays, and is usually required when a high amount of DNA is used to generate fragments for cloning. To determine the optimal Mg concentration for an assay, perform a Mg gradient PCR, during which the final Mg concentration in the reaction is increased in increments of 0.5 mM.
• Include PCR additives or enhancers in the reaction. Certain compounds, e.g. DMSO and betaine, are known to enhance the specificity and yield of problematic PCR reactions and can be included in the reaction at various concentrations. KAPA Enhancer 1, a proprietary additive supplied with KAPA2G Robust PCR Kits is recommended for use in problematic KAPA HiFi assays. Please note that PCR additives/enhancers may reduce the fidelity of KAPA HiFi.
• Check primer concentration and quality. Analyze primer stocks spectrophotometrically to confirm the purity and concentration of primers prior to use in KAPA HiFi reactions. Set reactions up with a final concentration of 0.3 µM of each primer. This should be sufficient for most applications; however, in rare cases the optimal primer concentration may have to be determined empirically. Always dilute and store primers in a buffered environment (e.g. 10 mM Tris-HC, pH 7.5 – 8.5) instead of in water. If KAPA HiFi yields good results with one primer pair but not another using the same template, the sequences of the nonfunctional primers may have to be rechecked to eliminate non-specific priming sites, or secondary structure formation. Resynthesis of primers may be required as primer quality often differs between vendors.
• Check template concentration and quality. Verify the integrity of template DNA by agarose gel electrophoresis and confirm the concentration and purity by spectrophotometric analysis. Use a control primer set to ensure that amplification is possible from the specific template. Perform a dilution series of template down to approximately 200 copies – sometimes inhibitors are present in a DNA preparation, which prevent amplification from “normal” amounts of template.
• Use KAPA HiFi GC Buffer instead of KAPA HiFi Fidelity Buffer. The GC Buffer may improve specificity and/or yield in troublesome assays. Re-optimization of cycling parameters (especially annealing temperature) might be needed when switching from Fidelity Buffer to GC Buffer.

10. Does KAPA HiFi enzyme produce blunt ends or fragments with 3’-dA-overhangs?
KAPA HiFi enzymes produce blunt-ended PCR products due to the 3’ – 5’ exonuclease activity that removes 3’-dA overhangs necessary for TA cloning. PCR products generated with KAPA HiFi enzymes are suitable for cloning using any commercially available blunt-end cloning kit. KAPA HiFi PCR products can also be prepared for cloning cloned into TA cloning vectors as follows:

• Purify the PCR product (spin columns are recommended) to remove the KAPA HiFi enzyme. If not fully removed, the enzyme will continue to remove 3’-dA overhangs added in subsequent steps!
• Set up a 50 μl A-tailing reaction containing the following: purified PCR product, 1 μl 10 mM dATP, 1 U (0.2 μl) KAPA Taq DNA Polymerase, 5 μl 10 x KAPA Taq Buffer A or B, and water up to 50 μl.
• Incubate for 10 min at 72 ºC
• Clone the 3’-dA-tailed KAPA HiFi PCR product according to the protocol supplied with the TA cloning kit.

11. Can I store KAPA HiFi PCR Kits at room temperature or 4 °C?
The recommended temperature for long-term storage of KAPA HiFi enzymes, KAPA HiFi Fidelity and GC Buffer, dNTPs and MgCl2 is -20 °C. However, these kit components or PCR master mixes prepared from them may be stored at 4 °C for short-term usage (up to a month). Don’t worry if you’ve left any component of the kit on your bench overnight or over the weekend – it will still work fine (but don’t make a habit of it!)

12. Can KAPA HiFi DNA Polymerase incorporate dUTP?
Like all B-family polymerases, KAPAHiFi DNA Polymerase is sensitive to dUTP poisoning and unable to extend past a dUTP present in a template strand. To minimize the negative impact of dUTP in KAPA HiFi reactions, always handle template DNA is carefully and use the minimum number of cycles. Only use high quality dNTPs supplied in KAPA HiFi PCR Kits.

13. Can PCR products generated with KAPA HiFi PCR Kits be analysed by dHPLC?
PCR products generated with KAPA HiFi or KAPA HiFi HotStart and KAPA HiFi Fidelity Buffer the recommended final concentrations, do not contain mineral oil, formamide, Proteinase K, BSA, high molecular weight stabilizers (e.g. PEG), detergents (e.g. SDS, Triton X-100, Tween 20, Nonidet-P40), glycerol, betaine or DMSO at final concentrations exceeding the maximum allowable concentrations for direct analysis using Transgenomic WAVE dHPLC systems.

PCR products generated with KAPA HiFi enzyme and KAPA HiFi GC Buffer must be diluted >2.5 times, or purified using a standard PCR cleanup kit, prior to analysis on Transgenomic WAVE dHPLC systems.