Molecular Methods and Computerized Analysis

Aitor Rementeria, Ana Belén Vivanco, Ainara Cadaval,

María Teresa Ruesga, Sonia Brena, José Pontón, Guillermo Quindós, and Javier Garaizar

1. Introduction

Infections caused by fungi (mycoses) are increasingly reported in many countries owing to greater life expectancy associated with an increase in quality of medical and surgical procedures, as well as the emergence of diseases or infections that affect the immune system such as AIDS. Nosocomial outbreaks of fungal infections are sometimes reported, and typing is then necessary to find the reservoirs, analyze the modes of transmission, study the antifungal susceptibility patterns, and investigate the susceptibility of the host (1,2).

In addition, the food industry is increasingly demanding typing methods that could help in selection of the best fungal strains, in order to incorporate them in the productive chains and augment the quality and security of food. This is the case for Saccha-romyces cerevisiae in the wine industry (3,4): the selection and characterization of indigenous or autochthonous strains is an important objective for the production of high-quality certified wines.

Several genotyping methods are now widely used for strain delineation of medically or economically important microorganisms belonging to the kingdom Fungi (5). Most molecular typing methods are comparable to those already described for bacteria, although the peculiarities of their nucleic acids increase the number of available methods (6-8). Although typing procedures based on the analysis of nucleic acid sequences have been developed (9), most genotyping methods currently in use are electrophoretically based, and the procedures include the visual comparison of nucleic acid band profiles or their reading with the help of computerized software.

Here we describe some of the most frequently used genotyping methods for fungi, based on polymerase chain reactions (PCR) (10-12), the isolation of chromosomal or mitochondrial DNA, and their restriction using endonuclease enzymes (1,8). The latter methods are exclusive for typing eukaryotic organisms and are based on the expected polymorphism obtained from the separation of large chromosomes using pulsed-field gel electrophoresis (PFGE) and the restriction of mitochondrial or chromosomal DNA. More sophisticated methods, such as those that combine endonuclease restriction with hybridization, are also available, although their use is less extensive and is limited mostly to research laboratories.

2. Materials

2.1. Randomly Amplified Polymorphic DNA Fingerprinting (RAPD-PCR)

1. Sabouraud dextrose agar with chloramphenicol (Pronadisa, Alcobendas, Madrid).

2. DNA extraction buffer: 2% v/v Triton X-100, 1% w/v sodium dodecyl sulfate (SDS), 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM Na2 EDTA. Sterilize by autoclaving and store at 4°C.

3. Phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v).

4. Glass beads. Sterilize by autoclaving.

5. Vortex.

6. 10X TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM Na2EDTA. Sterilize by autoclaving and store at 4°C.

7. Microfuge (Sanyo).

8. Absolute ethanol (Merck).

9. RNAse buffer: 1X TE buffer. Add 1 ^g/mL ribonuclease A (Sigma) prior to use.

11. Spectrophotometer (Kontron Instruments).

12. Ready-To-Go™ RAPD Analysis kit (Amersham Biosciences, Barcelona, Spain), providing room-temperature-stable beads containing buffer, PCR dNTPs, bovine serum albumin (BSA), AmpliTaq®, and Stoffel fragment and a set of PCR primers (Table 1).

13. Thermal cycler for PCR: Robocycler gradient 96 (Stratagene).

14. Tissue culture grade sterile water (Sigma).

15. Molecular grade agarose (Bio-Rad).

16. 10X TBE buffer: 890 mM Tris, 890 mM boric acid, 20 mM Na2 EDTA. Sterilize by autoclaving and store at room temperature.

17. pGEM DNA marker (Promega).

18. Gel loading solution (Sigma).

19. Standard apparatus for the electrophoresis of agarose gels and power supply (Bio-Rad).

2.2. Mitochondrial DNA Restriction Analysis (mtREA)

1. Yeast extract peptone dextrose (YPD) broth (Sigma).

2. Spheroplast buffer: 1 M sorbitol, 0.1 M EDTA, pH 7.5. Sterilize by autoclaving and store at 4°C.

3. 2.5 mg/mL Lyticase solution (Sigma).

4. Microfuge (Sanyo).

5. Resuspension buffer: 50 mM Tris-HCl, 20 mM EDTA, pH 7.4.

6. 10% w/v SDS. Sterilize by autoclaving and store at room temperature.

7. 5 M Potassium acetate. Sterilize by autoclaving and store at 4°C.

Table 1

Primers and PCR Conditions for Performing RAPD of Several Species of Fungi

Table 1

Primers and PCR Conditions for Performing RAPD of Several Species of Fungi

Fungi

Primers

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