A simplified procedure to analyse mitochondrial DNA from industrial yeasts.
A rapid method based on mtDNA restriction analysis is described for yeast strain identification. The method is an adaptation of that devised by Querol et a]. [Syst. Appl. Microbiol. 15 (1992) 439] for Saccharomyces cerevisiae wine strains, and consists of the standard miniprep isolation of yeast total DNA, and the use of restriction endonucleases that recognise a large number of sites in yeast nuclear DNA, but few sites in the mitochondrial DNA. In the adapted method, the propagation of yeast cells and restriction analysis were the steps mainly affected: cell growth was reduced to 36 h by using microfuge tubes, and the restriction analysis was carried out in just 33 min using a microwave oven for DNA digestion, and minigels for restriction fragment separation. The DNA extraction procedure was performed in the same way as in the original protocol. but slightly reducing the duration of each step and scaling down the volumes of the different solutions. enzymes and reagents used. As result, a large time reduction (52.5 h) was obtained compared to the original method. The DNA obtained can be directly digested with endonucleases displaying clear restriction patterns useful for S. cerevisiae yeast strain differentiation. In addition, strains belonging to other foodborne yeast species, including spoilage yeast species, can also be identified.
López
V
V
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot, Valencia, Spain.
Querol
A
A
Ramón
D
D
Fernández-Espinar
M T
MT
D004262:DNA Restriction Enzymes; D004272:DNA, Mitochondrial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D015151:Immunoblotting; D012150:Polymorphism, Restriction Fragment Length; D013997:Time Factors; D015003:Yeasts / Q000145:classification* / Q000235:genetics
D004272:DNA, Mitochondrial; D004262:DNA Restriction Enzymes