DNA isolation from small tissue samples using anion-exchange HPLC.
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 micrograms DNA were collected per injection. DNA was isolated from the oral tissue (264 micrograms), nasal mucosa (266 micrograms), esophagus (100 micrograms), pancreas (2 mg) and lymphocytes (5.8 micrograms/10(6) cells) of individual F344 rats. The DNA purity was high; it contained less than 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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Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595.
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification*; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D007202:Indicators and Reagents; D008214:Lymphocytes / Q000032:analysis; D008297:Male; D008745:Methylation; D008832:Microchemistry; D009602:Nitrosamines / Q000494:pharmacology; D009928:Organ Specificity; D051381:Rats; D011916:Rats, Inbred F344
D002273:Carcinogens; D007202:Indicators and Reagents; D009602:Nitrosamines; D006147:Guanine; C008450:7-methylguanine; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D004247:DNA; C008449:O-(6)-methylguanine