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.
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°C–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.
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.
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).
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.
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.
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.
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.