KAPA HiFi Uracil+

Take the fight out of bisulfite sequencing!

KAPA HiFi DNA Polymerase provides high fidelity next-generation sequencing library amplification with the highest yield, lowest bias, and most uniform sequence coverage available. These characteristics are retained in KAPA HiFi Uracil+ DNA Polymerase, a modified version of KAPA HiFi engineered to tolerate uracil residues. The new enzyme is particularly well suited for applications employing bisulfite DNA conversion, which typically produces low concentrations of AT-rich DNA.

KAPA HiFi HotStart Uracil+ ReadyMix (2X) is a ready-to-use cocktail containing all components required for PCR, except primers and template.

Amplifying NGS libraries? Go to our Library Amplification page.

 

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

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+ produced much higher yields (left) with very little size bias (right). Data on file.

Higher yields and reduced size bias

Replicate whole genome bisulfite-treated libraries were prepared from parasite gDNA, bisulfite-treated, and amplified using KAPA HiFi Uracil+ or Pfu Turbo Cx before paired-end sequencing on the Illumina GAIIx. KAPA HiFi Uracil+ resulted in higher overall coverage. [Data courtesy of the Wellcome Trust Sanger Institute, Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, and Institute for Molecular Medicine Finland (FIMM).] Data on file.

More uniform sequence coverage

Improved representation of AT-rich sequences

Applications
  • Methylation analysis – amplification of bisulfite-converted DNA for sequencing
  • Amplification of damaged DNA samples
  • Prevention of false-positive results due to carryover amplicon contamination
Kit Specifications and Contents / Storage

Store kits for 12 months at -20°C.

KAPA HiFi HotStart Uracil+ ReadyMix (2X) contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM).

Specifications

Spec
Description
Starting Material
DNA, cDNA, plasmid DNA bisulfite-treated DNA
Input Amount
1 - 100 ng for genomic DNA 10 pg - 1 ng for less-complex DNA
FAQs
What is the difference between HiFi Uracil+ and HiFi DNA Polymerase?

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

What are the key areas of optimization?

  • 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 to PCR-grade water, and minimize freeze-thaw cycles to limit degradation and maintain quality.
    • 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.
    • U DG initial incubation and deactivation: If your application requires prevention of carryover contamination, refer to the recommendations for UDG initial incubation and deactivation.

What are the standard cycling conditions for KAPA HiFi Uracil+ ReadyMix?

  • 3-step cycling profile (for optimal Ta in the range of 60–70°C ):
    • Initial denaturation: 2–5 minutes at 95°C
    • Denaturation: 20 seconds at 98°C
    • Annealing: 15 seconds at optimal Ta (60–70°C )
    • Extension: 30-60 sec/kb at 72°C
    • Final extension: 1 minutes at 72°C
    • Number of cycles: 15–40
  • 2-step cycling profile (for optimal Ta in the range of 68–75°C ):
    • Initial denaturation: 2–5 minutes at 95°C
    • Denaturation: 20 seconds at 98°C
    • Annealing/extension: 30 sec/kb at optimal Ta (68–75°C )
    • Final extension: 1 minutes at 72°C
    • Number of cycles: 15–40

What is the optimal annealing temperature for KAPA HiFi HotStart Uracil+ DNA Polymerase?

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°C–75°C and a combined annealing/extension time of 30 sec/kb may also be used.

Does magnesium need to be added to the PCR reaction with the KAPA HiFi HotStart Uracil+ ReadyMix?

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.

What does the HotStart formulation mean?

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.

What are the storage recommendations for KAPA HiFi HotStart Uracil+ ReadyMix?

The recommended temperature for long-term storage of KAPA HiFi HotStart Uracil+ ReadyMix 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 one month).

Why is KAPA HiFi HotStart Uracil+ ReadyMix well suited for NGS applications?

KAPA HiFi HotStart Uracil+ ReadyMix is especially well suited for NGS applications, providing reduced amplification bias and increased amplification efficiency of bisulfite-converted libraries. Library amplification with KAPA HiFi HotStart Uracil+ provides dramatic improvements in coverage depth uniformity and more representation across reference sequences.

Why is KAPA HiFi HotStart Uracil+ ReadyMix well suited for amplification of damaged DNA samples?

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 Uracil+ DNA Polymerase may allow high-fidelity amplification from damaged DNA templates containing uracil.

Why is KAPA HiFi HotStart Uracil+ ReadyMix well suited for use with the prevention of carryover amplicon contaminations?

KAPA HiFi 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 PCRs so that amplicons that may contaminate subsequent reactions are removed by digestion with UDG prior to amplification.

What is the optimal number of cycles for the amplification of bisulfite-converted NGS libraries?

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.

Ordering

KAPA HiFi HotStart Uracil+ ReadyMix (2X) contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM).

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK2802
07959079001
250 x 50 µL reactions
6.25 mL
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KK2801
07959052001
50 x 50 µL reactions
1.25 mL
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for pricing