State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China.
Plasmid. 2010 Nov;64(3):196-9. doi: 10.1016/j.plasmid.2010.06.005. Epub 2010 Jun 29.
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 λ 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.
在从酵母中提取总 DNA 的过程中，既能获得核 DNA 分子，也能获得线粒体 DNA（mtDNA）分子。在这里，我们描述了一种简单的酶法，使用 λ 外切核酸酶和 RecJ(f)的组合，通过从酵母细胞中分离的总 DNA 去除线性 DNA，从而获得纯的和完整的 mtDNA。这两种酶的组合能够有效地从光滑球拟酵母（Torulopsis）glabrata 的总 DNA 中去除线性 DNA，而 mtDNA 保持完整。通过 ARG1/2/5/8、URA3 和 COX1 的 PCR 扩增以及 RFLP 分析，分别检测 mtDNA 的纯度和完整性。该方法可用于 PCR 扩增或 RFLP 分析的 mtDNA 制备，而无需通过梯度超速离心或分步沉淀来纯化线粒体。该方法还成功应用于酿酒酵母、产朊假丝酵母、巴斯德毕赤酵母和解脂耶氏酵母等酵母属物种。
