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Nonenzymatic protocol for Pseudomonas aeruginosa DNA preparation and rapid subtyping by mini pulsed‐field gel electrophoresis
Author(s) -
LópezCánovas Lilia,
SánchezAlonso Axel,
Higginson David,
Ariosa Concepcion,
Clark Hilda,
Riverón Ana Maria
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390148
Subject(s) - subtyping , dna , electrophoresis , pseudomonas aeruginosa , agarose gel electrophoresis , lysis , gel electrophoresis , chromatography , agarose , chemistry , dna extraction , restriction enzyme , gel electrophoresis of nucleic acids , pulsed field gel electrophoresis , microbiology and biotechnology , bacteria , biology , biochemistry , polymerase chain reaction , genetics , genotype , gene , computer science , programming language
Abstract The fastest protocol for Pseudomonas aeruginosa subtyping by contour clamped homogeneous electric field (CHEF) electrophoresis takes around 20 h. It includes enzymatic sample preparation, DNA restriction and fragment separation. Here, P. aeruginosa cells embedded in agarose miniplugs were lysed and deproteinized by incubating the miniplugs for 30 min in a single nonenzymatic solution. DNA molecules were digested for 2 h with 5 U of Xba I, and fragments were separated in 4.96 h by miniCHEF electrophoresis at 10 V/cm. Total time for P. aeruginosa subtyping was 8 h. Control experiments included DNA preparation by enzymatic or nonenzymatic protocols, different times of DNA restriction and comparisons of DNA separations done by miniCHEF or CHEF electrophoresis. Both methods and chambers gave similar results, but the rapid nonenzymatic method and the miniCHEF gave them in less time. Cells grown in broth or on plates were useful for nonenzymatic DNA preparation. Thirteen P. aeruginosa isolates were successfully fingerprinted using the protocol described here.