Procedures: PCR Amplification, RFLP and Electrophoresis

 PCR Amplification

The primer pairs used to amplify the ITS (ITS1/ITS4) have been described by White et al. (1990). The oligonucleotide primers used to amplify the IGS were 5SA and CNL12 (Duchesne & Anderson, 1990; see Henrion et al., 1992 for the sequences). Primer CNL12 anneals at the 3' end of the 25S rRNA gene and is extended in the downstream direction whereas primer 5SA anneals at the 5' end of the 5S rDNA. The primers were synthesised and supplied by Bioprobe Systems (Montreuil-sous-Bois, France).

The PCR procedure for in vitro amplification of template DNA has been previously described in detail (Mullis & Faloona, 1987; Saiki et al., 1988).

• Reactants for amplification were combined in 0.5 ml polypropylene tubes to give a final volume of 100 microL and final concentration of: 10 mM-Tris-HCl (pH 8.8 at 25C), 50 mM-KCl, 1.5 mM-MgCl2, 20 pmol of each set of relevant unpurified primers, 1% Triton X100, 0.1% gelatine, 200 microM each of dATP, dCTP, dGTP, and dTTP, and 0.1 to 10 ng DNA template (50 microL of a 500- to 1000-fold dilution of DNA extracts).
• Following this, 2.5 units of Taq DNA polymerase (Bioprobe Systems) were added to the tube contents, which were then briefly mixed, centrifuged, and overlaid with two drops of mineral oil (Sigma Chimie) to prevent evaporation.
• The charged tubes were immediately placed in a Pharmacia-LKB GeneATAQ Controller.
• The thermal cycling parameters were an initial denaturation at 95C for 3 min, followed by 30 to 35 cycles of denaturation at 95C for 2 min, annealing at 50C for 25 sec, and extension at 72C for 2 min with a final extension at 72C for 10 min.
• Controls with no DNA were done in every series of amplification to test for the presence of contamination of reagents and reaction buffers.

 RFLP and Electrophoresis

• One tenth of the amplified DNA was digested 1 h with 5 to 10 units of various restriction enzymes (Alu I, Hinf I, Mbo I, Rsa I) (Pharmacia Fine Chemicals, BioLabs) according to the manufacturers' instructions.
• The amplification products were size-fractionated using a 1.5% composite agarose (0.5% wide-range agarose and 1.0% regular agarose) gel, whereas restriction fragments were separated using a 2% composite agarose (1.0% wide-range agarose and 1.0% regular agarose) gel (Sambrook et al., 1989).
• Agarose gels were stained with ethidium bromide, and photographed under UV light. fX174 DNA, digested with HaeIII, and the 100 Base-Pair Ladder were used as size markers.