Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption.
Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (10(8) and 10(7) CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 10(8) CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P < or = 0.005), 10(7) CFU of C. neoformans (P < or = 0.05), and 10(7) CFU of A. fumigatus (P < or = 0.01). Yields were within the same range for 10(8) CFU of C. neoformans and l0(7) CFU of C. albicans for both HSCD extraction and PC extraction. For 10(7) CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P < or = 0.05). Yields obtained from 10(8) and 10(7) CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.
Müller
F M
FM
Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. frank.mueller@mail.uni-wuerzburg.de
Werner
K E
KE
Kasai
M
M
Francesconi
A
A
Chanock
S J
SJ
Walsh
T J
TJ
D002725:Chloroform; D004271:DNA, Fungal / Q000302:isolation & purification*; D005658:Fungi / Q000235:genetics* / Q000254:growth & development; D003904:Mitosporic Fungi / Q000235:genetics*; D009172:Mycology / Q000379:methods*; D019800:Phenol; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D019105:Random Amplified Polymorphic DNA Technique; D014732:Vibration; D015003:Yeasts / Q000235:genetics*
D004271:DNA, Fungal; D019800:Phenol; D002725:Chloroform