A rapid method for the isolation of genomic DNA from Aspergillus fumigatus.
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.
Bir
N
N
Department of Zoology, University of Delhi, India.
Paliwal
A
A
Muralidhar
K
K
Reddy
P
P
Sarma
P U
PU
D001228:Aspergillosis / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000235:genetics*; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism; D004271:DNA, Fungal / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008872:Microwaves; D016133:Polymerase Chain Reaction
D004271:DNA, Fungal; D002686:Chitin; D002688:Chitinases