Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria.
We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.
Lehoux
D E
DE
Faculté de Médecine, Université Laval, Sainte-Foy, QC, Canada.
Sanschagrin
F
F
Levesque
R C
RC
D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics
D004251:DNA Transposable Elements; D009841:Oligonucleotides