Takara's Mutan-Super Express Km Kit is designed to achieve site-directed mutagenesis in just one day. It is based on Oligonucleotide-directed Dual Amber (ODA) method, using the advantages of LA (Long and Accurate) PCR.
Principle
Mutan-Super Express Km utilizes the pKF18k/19k vectors which contain dual amber mutations on the kanamycin-resistant gene. After a target sequence (< 2 kb) is inserted into either of these vectors, PCR is performed using an oligonucleotide containing the desired mutation and a selection primer to revert the amber mutations on the kanamycin-resistance genes.
The sequence between the two primers is then amplified, generating "Mutagenic-Selection DNA." Both strands of this amplified DNA fragment also serve as PCR primers, generating a nicked double-stranded plasmid. When this nicked DNA is transformed into E. coli MV1184 (sup0 strain), the nick is repaired. Transformants containing a desired site-specific mutation can be obtained after kanamycin selection. This simple procedure takes just one day and achieves mutation frequencies of greater than 80%.
