Separation of chromosomal DNA molecules fromC.albicansby pulsed field gel electropboresis

Abstract

Modifications have been made to standard pulse field gel eledrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of ekctrophoresls to 35°C. This enabled the largest SaccharomTces cerevlslat chromosome to be reprodbcibly resolved. More impressively, it enabled the DNA of Candida albkans to be clearly resolved Into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albkans chromosomes, which was estimated to be at least 5–10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highestmolecnlar weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albkans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes.