Plasmids with a kanamycin-resistance gene for site-directed mutagenesis using the oligodeoxyribonucleotide-directed dual amber method

Plasmid vectors carrying lacZ' and kanamycin-resistance (Km(R)) genes were constructed for site-directed mutagenesis (SDM) using the oligodeoxyribonucleotide (oligo)-directed dual amber (ODA) method [Hashimoto-Gotoh et al., Gene 152 (1995) 271-276]. The plasmids, designated pKF16k, pKF17k, pKF18k and pKF19k, correspond to the previously reported chloramphenicol resistant (Cm-R) ODA plasmids, pKF16c, pKF17c, pKF18c and pKF19c, respectively, but contain dual amber (am) codons in Km(R) instead of the Cm-R gene. The SDM procedure using the Km(R) ODA plasmids is essentially the same as that with Cm-R ODA plasmids, which utilizes two oligo primers for in vitro DNA synthesis, one (selection primer) for dual am reversions and the other (mutagenic primer) for the target site. The Km(R) ODA plasmids yield 5-10-times more DNA per culture volume as compared to the Cm-R ODA plasmids, and one can prepare selection agar medium simply by spreading Km solution on dried agar plate at a final concentration of 50-100 mu g/ml; due to the broad range of selecting antibiotic resistance.