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Rapid plasmid DNA isolation from mucoid gram-negative bacteria
P Domenico1, J L Marx, P E Schoch
1Infectious Disease Division, Winthrop-University Hospital, Mineola, Long Island, New York 11501.
Journal of Clinical Microbiology
|November 1, 1992
Summary
This study presents a new method for isolating plasmid DNA from gram-negative bacteria, overcoming challenges posed by exopolysaccharides. The technique ensures high-quality plasmid DNA suitable for characterization and analysis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Exopolysaccharides in gram-negative bacteria hinder plasmid DNA isolation and characterization.
- Conventional methods often yield insoluble or degraded plasmid DNA from encapsulated strains.
Purpose of the Study:
- To develop an efficient method for isolating and characterizing plasmid DNA from encapsulated gram-negative bacteria.
- To overcome limitations of existing plasmid DNA isolation techniques.
Main Methods:
- Bacteria cultured in bismuth nitrate and sodium salicylate to repress exopolysaccharide production.
- Mild acidic zwitterionic detergent extraction to remove surface contaminants.
- Alkaline detergent lysis for efficient cell disruption.
Main Results:
- New method yields plasmid DNA free of exopolysaccharides, with reduced lipopolysaccharide and protein contamination.
- Plasmids are highly soluble and exhibit clear banding patterns on agarose gels, allowing visualization of large plasmids (up to 180 kbp).
- Method effective for Klebsiella pneumoniae, Escherichia coli, Acinetobacter anitratus, Salmonella typhimurium, and Enterobacter species.
Conclusions:
- The developed method enables rapid, high-quality plasmid isolation and characterization from mucoid clinical isolates.
- This technique avoids organic solvents, CsCl gradients, and costly columns, offering a cost-effective alternative.
- The method is suitable for various gram-negative bacteria, excluding Pseudomonas aeruginosa due to bismuth inhibition.