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Two‐Dimensional Pulsed Field Minigel Electrophoresis with High Throughput Sample Format
Author(s) -
León Karen,
Riverón Ana María,
Arencibia Oscar,
Santamaría Yenis,
LópezCánovas Lilia
Publication year - 2013
Publication title -
journal of food safety
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 43
eISSN - 1745-4565
pISSN - 0149-6085
DOI - 10.1111/jfs.12042
Subject(s) - electrophoresis , pulsed field gel electrophoresis , gel electrophoresis , chromosome , dna , biology , microbiology and biotechnology , karyotype , chromatography , genetics , chemistry , gene , genotype
A rapid procedure for two‐dimensional pulsed field minigel electrophoresis (2 D ‐mini‐ PFGE ) with high throughput sample format was standardized to separate chromosomal DNA molecules of yeast. Molecules separation in the first dimension (1 D ) was done in 4.5 h using the contour‐clamped homogeneous electric field minichamber. 1 D ‐minigel was negatively stained with zinc‐imidazole and the lanes containing the chromosomal bands were excised from the 1 D ‐minigel. The strips were loaded into the second dimension (2 D ) minigel(s). 2 D runs were performed in the single or multiple minigels of the transversal alternating field electrophoresis ( TAFE ) or multi‐ TAFE minichamber, respectively, for 7 h. Total running time (1 D + 2 D ) was 11.5 h. The 2 D ‐mini‐ PFGE resolved co‐migrating molecules in the Saccharomyces cerevisiae 1 D ‐electrophoretic karyotypes and detected chromosome length polymorphisms in three distinct strains. Also, the multi‐ TAFE minichamber allowed obtaining up to 12 2 D ‐ DNA patterns simultaneously. This procedure could be suitable for monitoring of industrial yeast strains by molecular methods. Practical Applications This work reports the standardization of a rapid and economic two‐dimensional pulsed field minigel electrophoresis procedure with high throughput sample format to separate DNA molecules with chromosomal sizes. This procedure enhances the resolution of chromosomal DNA molecules in the 1 D ‐electrophoretic karyotypes from microorganisms and reveals chromosome length polymorphisms of different yeast strains without using restriction enzymes. The procedure also includes the staining of the 1 D ‐minigels with zinc‐imidazole. It avoids the environmental pollution caused by the ethidium bromide when the 1 D ‐ DNA molecules are visualized and prevents DNA damages due to UV ‐irradiation. The procedure permits to analyze from 1 to 12 DNA samples by 2 D ‐ PFGE simultaneously. It could be suitable for maintenance, monitoring and control of industrial yeast strains by molecular methods.