title: Nonenzymatic protocol for Pseudomonas aeruginosa DNA preparation and rapid subtyping by mini pulsed-field gel electrophoresis.
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 XbaI, 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.
authors.affiliation: Molecular Biology Department, Cuban Center for Neuroscience, National Center for Scientific Research, Havana, Cuba. llc@cneuro.edu.cu
authors.forename: Lilia
authors.initials: L
authors.lastname: López-Cánovas
authors.forename: Axel
authors.initials: A
authors.lastname: Sánchez-Alonso
authors.forename: David
authors.initials: D
authors.lastname: Higginson
authors.forename: Concepcion
authors.initials: C
authors.lastname: Ariosa
authors.forename: Hilda
authors.initials: H
authors.lastname: Clark
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Riverón
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000145:classification / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific