Robust PCR

The term “robustness” has many meanings and connotations. In the context of PCR, the most suitable definition is the capability (of a DNA polymerase or set of PCR reagents) to perform efficiently and consistently under a wide range of (sometimes unpredictable) conditions. Such conditions primarily refer to primer and template characteristics (such as length, GC content and secondary structure), the chemical environment (concentrations and purity of different components included in the reaction) and cycling parameters.

Wild-type Taq fails in many “difficult” assays, particularly those involving GC-rich, AT-rich or complex targets, or templates containing impurities or inhibitors. In contrast, KAPA2G Robust DNA Polymerase is a highly robust and versatile second-generation A-family DNA polymerase derived through a process of molecular evolution. The novel amino acid mutations in KAPA2G Robust offer higher processivity and specific activity, which translates to robust performance across a wide range of GC- and AT-rich templates and amplicons, as well as improved tolerance to common PCR inhibitors. In the HotStart formulation, the enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency.

KAPA2G Robust and KAPA2G Robust HotStart PCR Kits contain KAPA2G Buffers A and B and the proprietary additive, KAPAEnhancer 1, which offer extended optimization options for diverse and difficult templates. The kit also contains KAPA2G Robust HotStart GC Buffer, a novel buffer formulated specifically for GC-rich templates and amplicons.

KAPA2G Robust and KAPA2G Robust HotStart PCR Kits are recommended for all standard end-point PCR assays, particularly those in which wild-type Taq DNA polymerase does not perform satisfactorily. It is particularly suited for:

  • Amplification from templates with a high GC- or AT content.
  • Templates containing common PCR inhibitors (e.g. salts, urea, SDS and ethanol).
  • Amplification from crude samples, e.g. buccal swabs, cultured mammalian, yeast or bacterial cells (Colony PCR).
  • Optimization of low yield or low specificity assays.
  • High-throughput PCR using a single set of reaction and cycling parameters for multiple primer sets.

Amplicons generated with KAPA2G Robust (HotStart) are suitable for routine downstream applications, including restriction enzyme digestion, cloning and sequencing.