A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA from Animal Tissues and Cell Cultures

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Abstract
A two step procedure for the quantitative isolation of protein- and RNA-free double-strand DNA from animal tissue and cell homogenates is described. In the first step proteins not complexed with DNA are hydrolyzed with an immobilized protease (Proteinase K) that is separated by filtration after the de-proteinization. Then the DNA is adsorbed to hydroxylapatite (HA) and desorbed from the adsorbent by stepwise elution with buffers of increasing ionic strength. The DNA content was determined directly from the absorption at 260 nm. The melting curve of the isolated DNA showed that it was double stranded. The protein content in the DNA was determined from the ratio of the adsorbance at 260 to 230 nm. Non-histone proteins complexed to DNA determined the rate of deproteinization that was found to be tissue specific. These proteins were found to have a larger influence on the ratio A260/A230 than histones, indicating that their absorption (at 230 nm) is markedly perturbed when they are bound to DNA.