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KMID : 1161519990030010073
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Animal Cells and Systems
1999 Volume.3 No. 1 p.73 ~ p.78
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Improved procedure for large-scale isolation of mitochondrial DNA from mammalian tissues
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Hong Sung-Soo
Lee Chung-Choo
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Abstract
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Although there are several methods for the preparation of mitochondrial DNA (mtDNA) from mammalian tissues, most are relatively long ultracentri?fugation or manipulations by a small?scale method. We described a rapid method for large?scale extraction of mtDNA from human placental and horse liver tissues. The method is based on the preparation and homogenization of tissues, purification of crude mitochondria by differential centrifugations, and isolation of mtDNA by alkaline lysis. It was improved from pre?existing methods by replacing some steps with simpler ones and discarding many others. This method gives a high yield of pure mtDNA (approximately 1?5 mg from one placenta; ca. 400?600 g wet weight), depending on its sources (fresh tissue gave better results than frozen one). The resulting mtDNA indicated that this method can yield mtDNA in sufficient purity and quantity to identify the direct restriction analysis on agarose gel, random?primed labeling as a probe, and end labeling. Therefore, the method is ideal for obtaining good mtDNA samples to conduct routine restriction fragment length polymorphism (RFLP) analyses of natural populations for genetic studies
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KEYWORD
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Large-scale isolation, mtDNA, Alkaline lysis, Restriction analysis
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