Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption

Frank Michael C Müller, Katherine E. Werner, Miki Kasai, Andrea Francesconi, Stephen J. Chanock, Thomas J. Walsh

Research output: Contribution to journalArticle

Abstract

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 (108 and 107 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 108 CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P ≤ 0.005), 107 CFU of C. neoformans (P ≤ 0.05), and 107 CFU of A. fumigatus (P ≤ 0.01). Yields were within the same range for 108 CFU of C. neoformans and 107 CFU of C. albicans for both HSCD extraction and PC extraction. For 107 CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P ≤ 0.05). Yields obtained from 108 and 107 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.

Original languageEnglish (US)
Pages (from-to)1625-1629
Number of pages5
JournalJournal of Clinical Microbiology
Volume36
Issue number6
StatePublished - Jun 1998
Externally publishedYes

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Fungi
Chloroform
Phenol
Yeasts
DNA
Cryptococcus neoformans
Aspergillus fumigatus
Candida albicans
Pseudallescheria
Trichosporon
Rhizopus
Poisons
Fusarium
Restriction Fragment Length Polymorphisms
Cell Wall
Capsules
Saccharomyces cerevisiae
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Müller, F. M. C., Werner, K. E., Kasai, M., Francesconi, A., Chanock, S. J., & Walsh, T. J. (1998). Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. Journal of Clinical Microbiology, 36(6), 1625-1629.

Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. / Müller, Frank Michael C; Werner, Katherine E.; Kasai, Miki; Francesconi, Andrea; Chanock, Stephen J.; Walsh, Thomas J.

In: Journal of Clinical Microbiology, Vol. 36, No. 6, 06.1998, p. 1625-1629.

Research output: Contribution to journalArticle

Müller, FMC, Werner, KE, Kasai, M, Francesconi, A, Chanock, SJ & Walsh, TJ 1998, 'Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption', Journal of Clinical Microbiology, vol. 36, no. 6, pp. 1625-1629.
Müller FMC, Werner KE, Kasai M, Francesconi A, Chanock SJ, Walsh TJ. Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. Journal of Clinical Microbiology. 1998 Jun;36(6):1625-1629.
Müller, Frank Michael C ; Werner, Katherine E. ; Kasai, Miki ; Francesconi, Andrea ; Chanock, Stephen J. ; Walsh, Thomas J. / Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. In: Journal of Clinical Microbiology. 1998 ; Vol. 36, No. 6. pp. 1625-1629.
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abstract = "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 (108 and 107 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 108 CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P ≤ 0.005), 107 CFU of C. neoformans (P ≤ 0.05), and 107 CFU of A. fumigatus (P ≤ 0.01). Yields were within the same range for 108 CFU of C. neoformans and 107 CFU of C. albicans for both HSCD extraction and PC extraction. For 107 CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P ≤ 0.05). Yields obtained from 108 and 107 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.",
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