Open AccessTwo-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.
Author(s) -
Francis Barany
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.12.4202
Subject(s) - gene , pbr322 , genetics , biology , mutagenesis , plasmid , amp resistance , restriction site , tetracycline , mutation , microbiology and biotechnology , restriction enzyme , antibiotics
An efficient method for introducing two codons into a cloned gene has been applied to studying functional regions of the pBR322-encoded tetracycline-resistance gene and beta-lactamase (ampicillin-resistance) gene. Single-stranded hexameric linkers are inserted into a preexisting cohesive end restriction site to create a new (six-base recognition) restriction site. Insertion mutations are enriched by using biochemical selection or are selected by using a kanamycin-resistance cassette (biological selection). Phenotypes of insertion mutations isolated in the tetracycline-resistance gene support the hypothesis that it is comprised of two domains connected by a central hinge. Mutations in the beta-lactamase gene are temperature sensitive and demonstrate altered sensitivity to various beta-lactams and inhibitors.