Non-radioactive dot-blot DNA reassociation for unequivocal yeast identification.

Unequivocal and rapid classification of yeast cultures cannot be accomplished exclusively on the basis of unstable phenotypic traits, but requires molecular tests relating to the whole genome (or the largest possible portion of it). DNA-DNA reassociation meets this requirement, although many procedures proposed for calculating overall sequence similarity are expensive and time-consuming, thus restricting the possibility of unequivocal classification to a few specialized laboratories. A novel method, based on non-radioactive dot-blot hybridization of whole genomic DNA, has shown high and reproducible proportionality between the detected signal and the amount of double-stranded DNA effectively present on the membrane. This procedure has been optimized to obtain, within two working days, DNA relatedness values between unknown cultures and the type strains of the species previously indicated by a few conventional tests. The effective ability of the method to discriminate strains belonging to different species has been tested within taxonomic models consisting of yeast type cultures already certified by spectrophotometric reassociation.