A new method for isolating high purity DNA from small amounts of tissue is described. Tissue homogenates were treated with protease and extracted once with chloroform:isoamyl alcohol. The aqueous layer was injected onto a Waters Protein Pak anion-exchange HPLC column. Macromolecules were eluted in three fractions with NaCl in 20 mM sodium phosphate, pH 6.9, 5 M urea. The first fraction contained proteins and RNA, the second contained RNA and the third contained DNA. The DNA fraction was collected, desalted then lyophilized. Between 10 and 1000 μg DNA were collected per injection. DNA was isolated from the oral tissue (264 μg), nasal mucosa (266 μg), esophagus (100 μg), pancreas (2 mg) and lymphocytes (5.8 μg/106 cells) of individual F344 rats. The DNA purity was high; it contained <1% RNA or protein by weight. The methylation of DNA in the nasal mucosa of individual rats treated with 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was determined in DNA isolated by this method. The values determined agreed well with values determined from DNA isolated by precipitation methods from pooled tissues.
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The authors Wish to thank Chang-In Choi and the staff of the AHF Research Animal Faculty for providing the animal tissues, Rachel HeiMum for her excellent technical assistance in perfecting this method, Neil Trushin for providing chromatograms of DNA hydrolysates from DNA isolated by the modified Marmur method and Dietrich Hoffmann for his support. This study was supported by Research Grant RO1-CA-44161 from the National Cancer Institute.
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