A total of 150 Tunisian Cryptococcus isolates from clinical and environmental strains and the following standard strains representing each serotype of C. neoformans H99 (serotype A), JEC 21 (serotype D), the hybrid IHEM 13877 (serotype AD), C. gattii Wm 276 (serotype B) and IHEM 4159 (serotype C) were used.
Total genomic DNA was extracted from culture (108cells/ml) of Cryptococcus strains by means of the following 4 procedures: Protocol A used extraction with lyticase, phenol-chloroform and isoamylic alcohol; Protocol B used extraction with chelex, Protocol C used extraction using reagent kit (MasterPure yeast DNA purification KIT (Epicentre, Madison, USA)) and the new protocol D using urea chelex.
Protocol A: DNA extraction using lyticase, phenol-chloroform and isoamylic alcohol
The method for DNA extraction from culture by phenol-chloroform and isoamylic alcohol (Sigma-Aldrich, SP, Brazil) consisted of the suspension of cells recovered by centrifugation from 15 ml of a shaken (150 rpm) 18 h YEPD culture in 2 ml of SE (1.2 M sorbitol, o.1 M EDTA PH = 7.5) and following the method described by Shin-ichi [5
Protocol B: chelex DNA extraction
DNA was extracted using a rapid method based on thermal shock and the chelation of components other than nucleic acids by using a resin suspension, as previously described [6
Protocol C: Extraction by kit (MasterPure yeast DNA purification KIT (Epicentre, Madison, USA))
The DNA extraction with kit (MasterPure yeast DNA purification KIT (Epicentre, Madison, USA)) was performed according to manufacturer instructions.
Protocol D: new protocol using urea chelex
Procedure includes the following steps.
(i) Cells recovered by centrifugation from 15 ml of a shaken (150 rpm) 18 h YEPD culture were washed once with cold water, incubated 3 h in 2 ml of urea buffer (urea 8 M, NaCl 0.5 M, Tris 20 mM, EDTA 20 mM, SDS 2%, pH 8)
(ii) The pellet resuspended in 300 μl of distilled water in a microcentrifuge tube. A volume of 100 μl of Chelex solution (10% Chelex-100 [Bio-Rad, Hercules, Calif.] in an aqueous solution of 0.1% SDS, 1% Nonidet P-40, and 1% Tween 80) was added. The tubes were incubated at 95°C for 30 min and then on ice for 5 min. DNA was removed from the supernatant after 5 min of centrifugation (10,000 rpm) and stored at -20°C until used.
DNA sample concentrations were determined by spectrophotometry at the wave length of 260 nm for the DNA and 280 nm for proteins, and the purity observed using OD 260/OD 280, in NanoDrop equipment (Thermo Fisher Scientific, Wilmington, DE, USA). Concentration results are given in ng/μL, and the DNA purity results are reported as the OD 260/OD 280.
DNA quality determination
The DNA quality was accessed by electrophoresis and suitability for downstream application in RAPD analysis (Random Amplified polymorphic DNA). The quality of the DNA yielded by each method was determined by electrophoresing a 5 μl sample in a 0.8% TBE-agarose gel, stained with ethidium bromide. To further demonstrate the quality of the extracted DNA.
The RAPD analysis was carried out using primer six (5'-CCCGTCAGCA-3') in a volume of 50 μl. The following cycle conditions were used: initial denaturation at 95°C for five minutes, followed by 45 cycles of denaturation at 95°C for one minute, annealing at 36°C for one minute and amplification at 72°C for two minutes, and a final extension at 72°C for 10 minutes. Amplification products were separated by electrophoresis, on 2% agarose gels in 1 × TBE buffer at 150 V for 2.5 hours and stained with ethidium bromide and then visualized under UV light.