A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp.
The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.
Torres
R D
RD
Department of Microbiology and Medical Zoology, University of Puerto Rico School of Medicine, San Juan 00936-5067.
Oletta
C A
CA
Zlotnik
H
H
D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D009615:Nocardia / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes