• 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.