Takara's Mutan-Super Express Km site directed mutagenesis kit is based on ODA

method (Oligonucleotide-directed Dual Amber method) utilizing the advantage of LA PCR

technology and is designed to achieve site-directed mutagenesis just in one day.

The principle of the Mutan-Super Express Km is illustrated in Figure 1. Mutan-Super

Express Km utilizes the pKF18k/19k, same as Mutan-Express Km (Cat.# 6090) does.

Since the vector contains dual amber mutations on the kanamycin-resistant gene,

obtained transformants do not show kanamycin resistance when introduced in the sup

host strain, such as MV1184. After the target for mutagenesis is produced by cloning

into the pKF18k/19k vectors, PCR is performed using the oligonucleotide containing the

desired mutation and a selection primer to revert the amber mutations on the kanamycin-

resistant gene as PCR primers. With this PCR, the sequence between the two primers are

amplified, generating Mutagenic-Selection DNA. As this amplified DNA would be also

used as a PCR primer simultaneously, the polymerase reaction further progresses and

nicked double-stranded plasmid can be yielded. Utilizing the advantage of LA PCR

technology, Mutan-Super Express Km performs higher fidelity and longer PCR in

extension process of plasmid strands. When this nicked DNA is transformed into E. coli

MV1184 (sup strain), the nick is repaired and then transformants containing a desired

site-specific mutation can be grown on the medium containing kanamycin. Thus Mutan-

Super Express Km achieves the introduction of mutation at > 80% efficiency just in one

day by following a simple procedure.