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A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp
R D Torres1, C A Oletta, H Zlotnik
1Department of Microbiology and Medical Zoology, University of Puerto Rico School of Medicine, San Juan 00936-5067.
Abstract:
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.
Insights
A new method rapidly extracts undegraded genomic DNA from Nocardia species. This DNA is suitable for molecular diagnostics and characterization, overcoming previous limitations in Nocardia research.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Efficient DNA extraction is crucial for molecular manipulation and diagnostics of Nocardia species.
- Existing methods for Nocardia DNA extraction are often complex and inefficient, hindering research and clinical applications.
Purpose of the Study:
- To develop a simple, rapid, and efficient method for extracting undegraded genomic DNA from pathogenic Nocardia strains.
- To ensure the extracted DNA is suitable for downstream molecular applications like PCR and restriction digestion.
Main Methods:
- Culturing 14 Nocardia strains, treating with cycloserine, followed by cell lysis with lysozyme.
- Using proteinase K and sodium dodecyl sulfate for further lysis, then phenol-chloroform-isoamyl alcohol extraction.
- Precipitating DNA with isopropanol, RNase treatment, and analysis via agarose gel electrophoresis.
Main Results:
- Achieved average yields of 101 µg for Nocardia brasiliensis and 121 µg for Nocardia asteroides.
- The extracted genomic DNA was undegraded and suitable for restriction endonuclease digestion.
- The DNA was also effectively amplified using PCR, confirming its utility for diagnostic purposes.
Conclusions:
- The established method provides a reliable and efficient means for obtaining high-quality Nocardia genomic DNA.
- This advancement facilitates molecular characterization and diagnostic strategies for Nocardia infections.
- The method's simplicity and efficiency address a significant bottleneck in Nocardia research and clinical microbiology.