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Preparation of mitochondrial dna free of nuclear dna from animal tissues methylation level and pyrimidine nucleotide clustering degree as criteria for purification


, : Preparation of mitochondrial dna free of nuclear dna from animal tissues methylation level and pyrimidine nucleotide clustering degree as criteria for purification. Biokhimiya 41(1): 68-74

A method for preparing mitochondria DNA free of nuclear DNA and its fragments by treatment of mitochondria with DEAE-cellulose was developed. This method is based on binding nuclear nucleic acids and nucleoproteins to DEAE-cellulose particles in the media used for isolation of mitochondria. Treatment with DEAE-cellulose under the conditions described did not induce any visible degradation of mitochondria and mitochondrial DNA. The mitochondrial DNA preparations obtained from beef and rat liver are represented with closed circular molecules of contour length above 5.5 .mu. The 5-methylcytosine content in beef and rat mitochondrial DNA (3.03 and 2.0 mol%, respectively) is twice as much as in the corresponding nuclear DNA. Mitochondrial DNA strongly differs from nuclear DNA by having a lower degree of pyrimidine clustering; the amount of mono- and dipyrimidine fragments (about 32 mol%) in mitochondrial DNA is 1.5 times as large and the content of long pyrimidine clusters (hexa- and others) is 2-4 times as low as those in nuclear DNA. The methylation level and the pyrimidine clustering degree may be used as criteria for the purity of mitochondrial DNA from nuclear DNA.

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