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A rapid method for the isolation of genomic DNA from Aspergillus fumigatus
N Bir1, A Paliwal, K Muralidhar
1Department of Zoology, University of Delhi, India.
Abstract:
A majority of Aspergillus induced diseases are reported to be caused by Aspergillus fumigatus. In immunocompromized and post transplant cases it can lead to invasive aspergillosis. Due to this the molecular fingerprinting of aspergillus isolates by RFLP analysis and development of DNA diagnostic probes are gaining importance. Different methodologies are being adopted for extraction of the genomic DNA from fungus. The existing procedures for isolation of DNA are time consuming and range from several hours to few days. The most difficult step in the isolation of DNA from aspergillus species is to disrupt the tough chitin rich cell wall without causing damage to genomic DNA. We report here a rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase. The subsequent modification steps included are lysis and microwave treatment. The chromosomal DNA obtained by this procedure is 1.5-2.0 micrograms per mg of wet weight of mycelia and is observed to be minimally sheared. It is pure enough for restriction analysis and for use in the PCR to detect the gene coding for 18 kDa allergen which has been identified in our laboratory using western blot analysis with human patient sera.
Insights
This study presents a rapid DNA extraction method for Aspergillus fumigatus, crucial for diagnosing invasive aspergillosis. The new technique efficiently isolates pure genomic DNA for molecular fingerprinting and diagnostic probe development.
Area of Science:
- Mycology
- Molecular Biology
- Medical Diagnostics
Background:
- Aspergillus fumigatus causes significant diseases, particularly invasive aspergillosis in immunocompromised patients.
- Molecular fingerprinting and DNA diagnostic probes are vital for identifying Aspergillus isolates.
- Existing genomic DNA extraction methods are time-consuming and struggle with the fungus's chitin-rich cell wall.
Purpose of the Study:
- To develop a rapid and efficient method for extracting high-quality genomic DNA from Aspergillus species.
- To overcome the challenge of disrupting the tough fungal cell wall without damaging DNA.
- To provide DNA suitable for molecular analyses like RFLP and PCR.
Main Methods:
- Genomic DNA extraction utilizing enzymatic cleavage of chitin with chitinase.
- Incorporation of lysis and microwave treatment in the DNA isolation protocol.
- Assessment of DNA yield, purity, and integrity for downstream applications.
Main Results:
- A rapid DNA extraction protocol was successfully established.
- The method yields 1.5-2.0 micrograms of minimally sheared chromosomal DNA per mg of fungal mycelia.
- The extracted DNA is pure enough for restriction analysis and Polymerase Chain Reaction (PCR).
Conclusions:
- The novel chitinase-based method offers a significantly faster alternative for Aspergillus DNA extraction.
- This rapid DNA isolation is suitable for developing diagnostic tools and molecular fingerprinting of Aspergillus.
- The procedure facilitates the detection of specific genes, such as the one for the 18 kDa allergen.
