Site-directed mutagenesis frequently used in molecular biology, particularly in the study of protein structure-function relationships, gene expression and gene regulation. Many different strategies and methods for site-directed mutagenesis have been developed. The most commonly used methods employ complementary oligonucleotides to introduce mutations (additions, deletions or substitutions) at specific sites in a DNA fragment cloned into a plasmid. After hybridization of the oligonucleotides, which contain mismatched nucleotides at the mutation site, the entire plasmid is copied with a DNA polymerase. This generates nicked, circular DNA which is transformed into a suitable bacterial strain for nick repair and multiplication. Site-directed mutagenesis is often inefficient, resulting low yields of the mutant plasmid, a high background of parent DNA, as well as mutant plasmids containing spurious mutations, especially in cases where the total size of the recombinant plasmid exceeds 5 kb.
KAPA HiFi HotStart DNA polymerase is a novel, engineered B-family (proofreading) polymerase that is ideally suited for site-directed mutagenesis, for the following reasons:
- Superior fidelity. The error-rate of KAPA HiFi is approximately 100x lower than that of Taq and 30x lower than that of long-range enzyme blends that are often used for site-directed mutagenesis (especially when large plasmids are involved). With KAPA HiFi, clones containing spurious mutations are virtually eliminated.
- Superior amplification length and robustness. Unlike most B-family (proofreading) polymerases, KAPA HiFi is capable of amplifying plasmid targets up to 18 kb in size, with very little optimization of reaction conditions. This allows for true high fidelity site-mutagenesis of very large plasmids.
- Superior speed. KAPA HiFi DNA polymerase requires 15 – 30 sec/kb extension time per cycle. This is significantly faster than the 1 – 2 min/kb per cycle required by most other proofreading polymerases and long-range blends. The mutagenesis PCR may therefore be completed in a much shorter time, which increases turnaround time to mutants and limits template damage.