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Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.

F Barany
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Abstract

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.

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Amino Acid SequenceCodonDNA, RecombinantDNA, Single-StrandedDrug Resistance, MicrobialEscherichia coliGenetic EngineeringMutationOligodeoxyribonucleotidesOligonucleotidesPlasmidsTetracyclinebeta-LactamasesJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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F Barany. 1985. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides.. https://doi.org/10.1073/pnas.82.12.4202

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