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

Plasmid vectors carrying lacZ' and kanamycin-resistance (KmR) 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 (CmR) ODA plasmids, pKF16c, pKF17c, pKF18c and pKF19c, respectively, but contain dual amber (am) codons in KmR instead of the CmR gene. The SDM procedure using the KmR ODA plasmids is essentially the same as that with CmR 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 KmR ODA plasmids yield 5-10-times more DNA per culture volume as compared to the CmR 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 micrograms/ml; due to the broad range of selecting antibiotic resistance.