Molecular analysis for rapid extraction of genomic DNA from medically important yeasts

ABSTRACT

Current methods of DNA extraction from four different pathogenic yeasts are often time-consuming and require the use of toxic and carcinogenic chemicals. DNA isolation from yeast 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 four medically important yeats [Saccharomyces cerevisiae, Candida utilis, Cryptococcus neoformans and Trichosporon beigelii]. Two different inocula [10[7] and 10[8] CFU] were compared for optimization of obtained yields. The entire extraction procedure was performed on as four samples within one hour compared to six hours for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 108 CFU of C. utilis, T. beigelii [P S. cerevisiae [P methods. The rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism RFLP, polymerase chain reaction PCR. We concluded that mechanical disruption of yeast cells by HSCD is a safe, rapid and efficient procedure for extracting genomic DNA from medically important yeasts