The PCR amplification of GC-rich DNA is often problematic due to stable secondary structures in the DNA that are resistant to melting. These secondary structures cause DNA polymerases to stall, resulting in incomplete and non-specific amplification. Many different methods and additives have been developed to facilitate template denaturation. Successful amplification with wild-type polymerases is very dependent on the assay, GC content and the complexity of the template DNA.
KAPA2G Robust DNA Polymerase, a second-generation DNA polymerase derived from wild-type Taq through molecular evolution, and KAPA HiFi DNA Polymerase, an engineered, high fidelity B-family DNA polymerase, are both ideally suited for the amplification of GC-rich DNA. The unique properties of these enzymes include high processivity and improved tolerance to DNA melting agents. Both enzymes are supplied with proprietary reaction buffers developed specifically for GC-rich PCR and allow for the efficient amplification of GC-rich DNA using extension times of 15 – 30 sec/kb per cycle. Both enzymes are available in antibody-mediated HotStart formulations.
KAPA2G Robust and/or KAPA HiFi offer comprehensive solution(s) for GC-rich PCR, including the amplification of DNA sequences:
- with an extremely high content (>80%)
- containing GC clusters
- using primers with a high GC content
- with an average overall GC content, but stable secondary structure