Abstract
Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray.
Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20–70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa.
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Acknowledgments
PFGE equipment and protocols have initially been set up by Dietmar Grothues, Wilfried Bautsch, Joachim Greipel, Uta Koopmann, and Ute Römling. The protocols were subsequently modified, evaluated, and adapted to the needs of individual applications by (in chronological order) Ute Römling, Rainer Fislage, Thomas Heuer, Jutta Ulrich, Christiane Bürger, Karen Larbig, Stefanie Breitenstein, Peter Gudowius, Christian Weinel, and Jens Klockgether. The protocol for genotyping of P. aeruginosa by oligonucleotide microarray has been developed by Lutz Wiehlmann in collaboration with a team at CLONDIAG Chip Technologies GmbH.
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Tümmler, B. (2014). Genotyping Methods. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_5
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DOI: https://doi.org/10.1007/978-1-4939-0473-0_5
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