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Biotechniques. 1999 Mar;26(3):473-8, 480.

Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria.

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1
Faculté de Médecine, Université Laval, Sainte-Foy, QC, Canada.

Abstract

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.

PMID:
10090989
DOI:
10.2144/99263st02
[Indexed for MEDLINE]
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