Comparative mapping of the Pseudomonas aeruginosa PAO genome with rare‐cutter linking clones or two‐dimensional pulsed‐field gel electrophoresis protocols

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Abstract
The Spe1 map of the Pseudomonas aeruginosa PAO (DSM 1707) chromosome was constructed by utilizing two‐dimensional pulsed‐field gel electrophoresis (PFGE) and rare‐cutter linking clones. After end‐labeling and fluorescence staining of macrorestriction fragments had been combined, the two‐dimensional PFGE analyses of partial‐total digests and reciprocal double digests were sufficient for the placement of all fragments on the genomic map. Spe1 linking fragments were isolated from BamH1, Pst1, and EcoR1 genomic libraries of P. aeruginosa PAO. After separation of the heterogeneously sized populations of Spe1‐linearized and uncut circular plasmid DNAs by field inversion polyacrylamide gel electrophoresis, the gel‐eluted linear DNAs were recircularized and subcloned. The 46 analyzed Spe1 linking clones recognized 16 of the 38 fragment links on the Spe1 genome map of P. aeruginosa PAO. The alignment with linking clones was consistent with that obtained from two‐dimensional PFGE mapping protocols.