Nonenzymatic protocol for Pseudomonas aeruginosa DNA preparation and rapid subtyping by mini pulsed-field gel electrophoresis.
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.
López-Cánovas
Lilia
L
Molecular Biology Department, Cuban Center for Neuroscience, National Center for Scientific Research, Havana, Cuba. llc@cneuro.edu.cu
Sánchez-Alonso
Axel
A
Higginson
David
D
Ariosa
Concepcion
C
Clark
Hilda
H
Riverón
Ana Maria
AM
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
D004269:DNA, Bacterial; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific