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KAPA HiFi Uracil+ Kit

For Research Use Only. Not for use in diagnostic procedures.
KAPA HiFi Uracil+ Kit

Overview
 

The KAPA HiFi Uracil+ Kit forms part of the family of KAPA HiFi Kits, where KAPA HiFi HotStart Uracil+ DNA Polymerase is a modified version of KAPA HiFi HotStart DNA Polymerase that is engineered to tolerate uracil residues in bisulfite-treated DNA. Bisulfite treatment of DNA results in the conversion of unmethylated cytosine residues into uracil, while the methylated residues are left unmodified. Enzymes used for library amplification in bisulfite-sequencing workflows must be able to read through uracil residues and tolerate low concentrations of AT-rich DNA. Traditional high-fidelity DNA polymerases are typically not suitable for this type of application as the enzymes stall when a uracil is encountered.

KAPA HiFi HotStart Uracil+ DNA Polymerase has been developed to read through uracil residues and other modified bases, while still retaining the performance benefits of KAPA HiFi HotStart DNA Polymerase, including ultra-high fidelity, high efficiency and low-bias amplification. This results in lower duplication rates and improved coverage of challenging regions.

Benefits of KAPA HiFi Uracil+ Kits

  • Higher and more uniform bisulfite-sequencing coverage for templates containing uracil compared to other methods
  • Higher yields, low duplication rates and fewer wasted sequencing reads
  • Convenience with the consistency of one core enzyme
  • Trusted as shown through over 10 years in the industry as well as thousands of peer-reviewed publications

Product highlights

Amplification of uracil-containing template

  • Effective amplification through uracil and other modified residues

 

View data on KAPA HiFi Uracil showing no inhibition by dUTP.

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Figure 1. KAPA HiFi Uracil+ is not inhibited by dUTP. SYBR Green real-time PCR was used to monitor amplification of a 452 bp amplicon over ten-fold dilutions (80 pM – 0.8 fM) with or without 0.2 mM dUTP. KAPA HiFi DNA Polymerase shows typical inhibition by uracil, while KAPA HiFi Uracil+ DNA Polymerase shows no amplification inhibition.

Figure 1. KAPA HiFi Uracil+ is not inhibited by dUTP

Figure 2. KAPA HiFi Uracil+ is not inhibited by dUTP. SYBR Green real-time PCR was used to monitor amplification of a 452 bp amplicon over ten-fold dilutions (80 pM – 0.8 fM) with or without 0.2 mM dUTP. KAPA HiFi DNA Polymerase shows typical inhibition by uracil, while KAPA HiFi Uracil+ DNA Polymerase shows no amplification inhibition.

Figure 2. KAPA HiFi Uracil+ is not inhibited by dUTP
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High-efficiency, low bias amplification

  • Reduced cycles and lower PCR duplicates through high-efficiency amplification of bisulfite-converted libraries. 
  • Low bias amplification results in improved representation of all library fragments, regardless of size. 

 

View comparative data showing higher yield and less size bias using KAPA HiFi Uracil+ compared to Agilent Pfu Turbo Cx.

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Figure 3. KAPA HiFi Uracil+ provides higher yields and minimal size bias. Human whole genome bisulfite-treated libraries were amplified using standard protocols with 12, 14, or 16 cycles and the amplified libraries were analyzed using a Bioanalyzer 2100 High Sensitivity DNA chip. When compared with Agilent Pfu Turbo Cx, KAPA HiFi Uracil+ DNA Polymerase produced much higher yields (left) with very little size bias (right)

Figure 3 .KAPA HiFi Uracil

Figure 4. KAPA HiFi Uracil+ provides higher yields and minimal size bias. Human whole genome bisulfite-treated libraries were amplified using standard protocols with 12, 14, or 16 cycles and the amplified libraries were analyzed using a Bioanalyzer 2100 High Sensitivity DNA chip. When compared with Agilent Pfu Turbo Cx, KAPA HiFi Uracil+ DNA Polymerase produced much higher yields (left) with very little size bias (right)

Figure 4. KAPA HiFi Uracil
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Higher and more uniform coverage of bisulfite-convert libraries

  • Improved representation of AT-rich and other difficult regions
  • More even coverage leads to higher coverage depth uniformity

 

See data displaying higher and more uniform coverage with whole genome bisulfite (WGBS) libraries.

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Figure 5. Higher and more uniform coverage leads to improved representation of AT-rich sequences with KAPA HiFi HotStart Uracil+ DNA Polymerase compared to Agilent Pfu Turbo Cx kit. 100 ng of bisulfite-treated library DNA was amplified (8 cycles) with the KAPA HiFi HotStart Uracil+ ReadyMix Kit or Agilent Pfu Turbo Cx.

Figure 5. Higher and more uniform coverage

Figure 6. Higher and more uniform coverage leads to improved representation of AT-rich sequences with KAPA HiFi HotStart Uracil+ DNA Polymerase compared to Agilent Pfu Turbo Cx kit. 100 ng of bisulfite-treated library DNA was amplified (8 cycles) with the KAPA HiFi HotStart Uracil+ ReadyMix Kit or Agilent Pfu Turbo Cx.

Figure 6. Higher and more uniform coverage
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*Data on file.

 

Roche Sequencing Solutions offers KAPA HiFi Uracil+ Kits, containing the engineered KAPA HiFi HotStart Uracil+ DNA Polymerase enzyme, formulated in a Readymix format. These kits form part of the family of KAPA HiFi Kits, offering the same benefits, but evolved for the amplification of uracil and modified residues.

The table below describes the specifications for the KAPA HiFi HotStart Uracil+ ReadyMix Kits:

KAPA HiFi HotStart Uracil+ ReadyMix Kit

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KAPA HiFi HotStart Uracil+ ReadyMix Kit

   
HotStart Enzyme

Yes
Format

ReadyMix
Kit Contents KAPA HiFi HotStart Uracil+ ReadyMix
Recommended Applications

PCR and NGS applications requiring amplification of uracil or modified residues e.g. bisulphite-converted library amplification
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KAPA HiFi HotStart Uracil+ ReadyMix (2X) contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM)

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Kit Code Roche Cat. No Description Kit Size
KK2801 07959052001 KAPA HiFi HotStart Uracil+ ReadyMix Kit (50 rxn) 50 x 50 µL reactions
KK2802 07959079001 KAPA HiFi HotStart Uracil+ ReadyMix Kit (250 rnx) 250 x 50 µL reactions
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Frequently asked questions

  • Methylation analysis, amplification of bisulfite-converted DNA for PCR or NGS applications
  • Amplification of damaged DNA samples
  • Prevention of false-positive results due to carryover amplicon contamination

The uracil-binding pocket of KAPA HiFi HotStart DNA Polymerase has been inactivated, enabling amplification of uracil-containing DNA, thus creating KAPA HiFi HotStart Uracil+ DNA Polymerase. KAPA HiFi HotStart Uracil+ DNA Polymerase exhibits the same high-efficiency, low-bias amplification and coverage uniformity as the unmodified enzyme.

Reaction set-up:

  • Amount of starting template: Use 1 – 100 ng for genomic DNA and 10 pg – 1 ng for less complex DNA.
  • Quality of template: While KAPA HiFi HotStart Uracil+ ReadyMix tolerates uracil, deamination of dCMP to dUMP in the DNA template will generate G/C to A/T mutations during amplification. Always dilute and store DNA in a buffered solution (e.g. TE or Tris-HCl, pH 8.0–8.5) instead of PCR-grade water.
  • 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.
  • dUTP and UDG concentrations: If your application requires prevention of carryover contamination, refer to the manufacturer’s recommendations for dUTP and UDG concentrations.


Cycling parameters:

  • Initial denaturation: An initial denaturation time of 2 – 5 minutes at 95°C is recommended to ensure full denaturation. Use 5 minutes for complex, genomic DNA and/or GC-rich targets and at least 45 seconds for less complex templates.
  • Denaturation time during cycling: To ensure adequate template denaturation, always denature for 20 seconds at 98°C in each PCR cycle. For templates with abundant priming sites, such as purified vector DNA or NGS libraries, shorter denaturation times of 15 seconds can be used.
  • Extension time: For amplicons ≤1 kb, use 15 seconds per cycle. For long targets or to improve yields, use 30 – 60 sec/kb per cycle.
  • Annealing temperature: The optimal annealing temperature for a specific primer pair is likely to be higher than when used in a conventional PCR buffer. Start with an annealing temperature of 60°C . If non-specific products are obtained, determine the optimal annealing temperature in an annealing temperature gradient PCR (60 – 75°C). A 2-step protocol with a combined annealing/extension step at 68 – 75°C may be used for 30 sec/kb.
  • Cycle number: ≤25 cycles are recommended for most high fidelity applications. Reactions with low template concentrations may require 30 – 35 cycles.

The recommended extension rate is 15 seconds for targets ≤1 kb and 30 – 60 sec/kb for long targets, or to improve yields.

The optimal annealing temperature for a specific primer pair is likely to be higher than when used in a conventional PCR buffer. An annealing temperature of 60°C is recommended as a starting point. Two-step cycling protocols, with a combined annealing/extension temperature in the range of 68 – 75°C and a combined annealing/extension time of 30 sec/kb may also be used.

KAPA HiFi HotStart Uracil+ ReadyMix contains Mg2+ at a 1X concentration of 2.5 mM, which is optimal for most applications. Reactions may be supplemented with any PCR-grade MgCl2 solution. Add 0.5 µL of a 25 mM MgCl2 solution to increase the final concentration in a 50 µL reaction by 0.25 mM. It may be necessary to test a range of concentrations to determine the optimal conditions for your specific PCR or NGS application.

A proprietary antibody inactivates the polymerase until the first cycle of thermal denaturation. This minimizes spurious amplification products that may result from non-specific priming events during reaction setup and initiation and increases overall reaction efficiency.

Upon receipt, store the entire kit at -20°C in a constant-temperature freezer. Kits may be stored at 4°C for regular, short-term use (up to one month). Provided that it has been handled carefully and not contaminated, the product is not expected to be compromised if left at room temperature for short periods of time (up to three days). Long-term storage at room temperature or 4°C is not recommended. Please note that reagents stored above -20°C are more prone to degradation when contaminated by the user; storage at such temperatures is therefore at the user’s own risk.

KAPA HiFi HotStart Uracil+ ReadyMix Kits are especially well suited for NGS applications, providing high-efficiency, low-bias amplification of bisulfite-converted libraries. Library amplification with KAPA HiFi HotStart Uracil+ DNA Polymerase provides improvements in coverage depth uniformity and more representation across reference sequences.

Cytosine deamination occurs spontaneously over long periods of time, and more rapidly at elevated temperatures, and results in the accumulation of uracil in DNA and among free nucleotides. When other proofreading enzymes fail, KAPA HiFi HotStart Uracil+ DNA Polymerase allows high-fidelity amplification from damaged DNA templates containing uracil.

KAPA HiFi HotStart Uracil+ DNA Polymerase readily incorporates dUTP during amplification, and can therefore be used in conjunction with uracil-DNA-glycosylase (UDG) to prevent carryover contamination. dUTP is added to PCR reactions so that amplicons that may contaminate subsequent reactions are removed by digestion with UDG prior to amplification.

It is important to optimize the cycle number for your specific samples and protocol; 8–14 cycles of PCR are usually required for sufficient amplification of bisulfite-converted NGS libraries. As with all NGS applications, optimal library amplification should provide sufficient material for sample validation (QC) and sequencing, while avoiding excessive amplification which may result in undesirable artifacts such as PCR duplicates, amplification bias, reduced library complexity, and PCR errors.

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