Method to purify mitochondrial DNA directly from yeast total DNA

During the purification of total DNA from yeast, both nuclear and mitochondrial DNA (mtDNA) molecules are obtained. Here, we describe a simple enzymatic method using a combination of exonuclease and RecJ(f) to obtain pure and intact mtDNA by removing linear DNA from total DNA isolated from yeast cells. The combination of the two enzymes efficiently removed linear DNA from the total DNA of Candida (Torulopsis) glabrata, leaving the mtDNA intact. The purity and integrity of mtDNA was assayed by PCR amplification of ARG1/2/5/8, URA3 and COX1, and by RFLP analysis, respectively. This method can be used to prepare mtDNA for PCR amplification or RFLP analysis without the need for purification of mitochondria by gradient ultracentrifugation or fractional precipitation. The method was also successfully applied to the yeast species Saccharomyces cerevisiae, Candida utilis, Pichia pastoris and Yarrowia lypolytica. (C) 2010 Elsevier Inc. All rights reserved.