pcR Amplification of Rapidly ExtractedDNA from Different Pathogenic Yeasts

DNA extraction from 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 chao tropic reagents and lysing matrices in composition to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from four medically important yeasts (Saccharomyces cerevisiae, Candia utilis, Cryptococcus neogormens and Trichosporon beige/ill Two different inocula (107 and 108 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. beigelli (P 0.005),107 CFU of C. neoformans and S. cerevisiae (P ~ 0.05) yields were within the same range of 108 CFU of C. neogormans and 107 CFU of C. utilis 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, 108 were significantly greater by the HSCD extraction than PC 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