Two‐Dimensional Pulsed Field Minigel Electrophoresis with High Throughput Sample Format

Abstract

AbstractA rapid procedure for two‐dimensional pulsed field minigel electrophoresis (2D‐mini‐PFGE) with high throughput sample format was standardized to separate chromosomal DNA molecules of yeast. Molecules separation in the first dimension (1D) was done in 4.5 h using the contour‐clamped homogeneous electric field minichamber. 1D‐minigel was negatively stained with zinc‐imidazole and the lanes containing the chromosomal bands were excised from the 1D‐minigel. The strips were loaded into the second dimension (2D) minigel(s). 2D 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 (1D + 2D) was 11.5 h. The 2D‐mini‐PFGE resolved co‐migrating molecules in the Saccharomyces cerevisiae 1D‐electrophoretic karyotypes and detected chromosome length polymorphisms in three distinct strains. Also, the multi‐TAFE minichamber allowed obtaining up to 12 2D‐DNA patterns simultaneously. This procedure could be suitable for monitoring of industrial yeast strains by molecular methods.Practical ApplicationsThis 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 1D‐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 1D‐minigels with zinc‐imidazole. It avoids the environmental pollution caused by the ethidium bromide when the 1D‐DNA molecules are visualized and prevents DNA damages due to UV‐irradiation. The procedure permits to analyze from 1 to 12 DNA samples by 2D‐PFGE simultaneously. It could be suitable for maintenance, monitoring and control of industrial yeast strains by molecular methods.