| Popis: |
Literature data describe different available methods and commercial tests for fungal genomic DNA isolation, with various DNA yields and quality. Tests are usually designed for general DNA isolation, with different procedures for different types of cells, while the methodology is often applicable only to specific species, and does not give good results if used for specific filamentous fungal species. The aim of the study was to determine an optimal method for isolation of DNA from Aspergillus flavus (NRRL 3251) with best integrity, purity and quantity, to be used for PCR analysis. Five different methods with some modifications were compared. All modifications were made to adjust desired growth conditions in liquid medium (GMS) and expected amount of mycelium for analysis (0.1 – 1 g of wet matter). After incubation, mycelium was stored at -20°C until DNA isolation. Examined methods were: Pure PCR template preparation kit (Roche), Rapid mini-preparation of fungal DNA for PCR (Liu et al., J Clin Microbiol 38, 2000: 471), the CTAB method, Isolation of genomic DNA for insects (Qiagen) and Method for microbial DNA extraction from soil for PCR amplification (Yeates et al., Biol Proced Online 1, 1998: 40). Following isolation, DNA was stored at -20°C before further analysis. DNA quantification and purity estimation were performed by NanoDrop ND 1000 spectrophotometer and the results were compared to Perkin Elmer Precisely Lambda 25 UV/VIS spectrophotometer. DNA integrity and quantity were determined by gel-electrophoresis. Despite claimed as suitable for fungal DNA isolation, a few of the methods did not give satisfactory results, ranging from great amounts of impure DNA with high integrity to small amounts of pure DNA with low integrity. DNA concentrations varied from 22.58 ± 2.04 ng/L to 5507.07 ± 45.96 ng/L with A260/280 from 1.1 to 2.2 using nanodrop or 1749.00 ± 160.58 ng/L to 14870.00 ± 112.38 ng/L with A260/280 from 0.80 to 2.27 using conventional spectrophotometer. Optimal results for further PCR amplification were obtained with modified Yeates et al. method, which gave 5480.13±52.45 ng/L DNA of very good integrity but low purity (A260/280 = 1.17). |
