An optimized protocol for the production of interdelta markers in Saccharomyces cerevisiae by using capillary electrophoresis.
The amplification of genomic sequence blocks flanked by delta elements of retrotransposon origin has proved to be a very convenient method for molecular characterization of Saccharomyces cerevisiae strains. Fluorescent automated capillary electrophoresis (CE) was used to detect interdelta marker (IDM) patterns in S. cerevisiae, using the ABI Prism 3130 Genetic Analyzer. Main experimental parameters were studied and the optimal conditions for IDM amplification and samples run on the CE apparatus were determined. Fingerprints from fluorescent-labelled IDM produced using CE with the same sample analyzed by agarose electrophoresis (AE) were compared. The CE analysis was able to distinguish 43 different IDM profiles among 45 S. cerevisiae isolates with a discriminating capacity of 99.8%, whereas the AE analysis of the same samples allowed the identification of 27 different patterns (discriminatory power equal to 96%). Detection of fluorescent IDM was fast and reliable, and it facilitated data comparison. For the first time in our knowledge, the fluorescent CE proved to be well suited for IDM fingerprinting. Moreover, it could be routinely applied for the molecular differentiation of S. cerevisiae strains.
Tristezza
Mariana
M
C.N.R.-Institute of Sciences of Food Production (ISPA), Operative Unit of Lecce, via Provinciale Lecce-Monteroni, 73100 Lecce, Italy.
Gerardi
Carmela
C
Logrieco
Antonio
A
Grieco
Francesco
F
D016172:DNA Fingerprinting / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005819:Genetic Markers; D016533:Mycological Typing Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013194:Staining and Labeling / Q000379:methods
D004271:DNA, Fungal; D005819:Genetic Markers