High Fidelity PCR

PCR is used either for detection of specific sequences, or to generate DNA for downstream applications such as sequencing, cloning, site-directed mutagenesis, gene assembly or protein expression. While Taq polymerase and second-generation A-family polymerases such as KAPA2G Robust and KAPA2G Fast allow reliable and sensitive detection of sequences, these enzymes have relatively high error-rates (2.4 x 10E-5). In addition, A-family DNA polymerases are unable to synthesize long amplicons, because extension from mismatches occurs inefficiently. Error-free, full-length gene sequences for downstream applications are therefore not achievable with A-family polymerases.

Wild-type, high fidelity B-family polymerases are capable of error rates ~20-fold lower than Taq as a result of their 3’ –5’ exonuclease or proofreading activity. However, many B-family enzymes, such as Pfu, Vent, or KOD, are notorious for low sensitivity, poor priming specificity, long extension times and low yield.

KAPA HiFi DNA Polymerase is an engineered B-family polymerase that overcomes the limitations of first-generation high fidelity PCR technology. The unique KAPA HiFi DNA polymerase, reaction buffers and protocols set new standards in sensitive, specific and fast PCR amplification of full-length genes with very low error rates. KAPA HiFi, which is also available in an antibody-mediated HotStart formulation, offers the following advantages for high fidelity PCR:

  • The lowest error-rate available for PCR – 2.8 x 10E7 errors/nucleotide incorporated (100x higher fidelity than Taq polymerase).
  • Amplification of up to 15 kb from genomic DNA, or 18 kb from plasmid DNA.
  • Two reaction buffers enable optimisation for challenging templates such as those with a high GC content.
  • Short extension times (30 sec/kb per cycle) significantly reduce total cycling time.