A simplified procedure to analyse mitochondrial DNA from industrial yeasts

A rapid method based on mtDNA restriction analysis is described for yeast s train identification. The method is an adaptation of that devised by Querol et al. [Syst. Appl. Microbiol. 15 (1992) 439] for Saccharomyces cerevisiae wine strains, and consists of the standard miniprep isolation of yeast tot al DNA, and the use of restriction endonucleases that recognise, a large nu mber of sites in yeast nuclear DNA, but few sites in the mitochondrial DNA. In the adapted method, the propagation of yeast cells and restriction anal ysis were the steps mainly affected: cell growth was reduced to 36 h by usi ng 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 sam e way as in the original protocol, but slightly reducing the duration of ea ch step and scaling down the volumes of the different solutions, enzymes an d reagents used. As result, a large time reduction (52.5 h) was obtained co mpared to the original method. The DNA obtained can be directly digested wi th endonucleases displaying clear restriction patterns useful for S. cerevi siae yeast strain differentiation. In addition, strains belonging to other foodborne yeast species, including spoilage yeast species, can also be iden tified. (C) 2001 Elsevier Science B.V. All rights reserved.