The diaminobenzoic acid (DABA) reaction with DNA, first described by Kissane and Robbins (J. M. Kissane and E. Robbins, 1958, J. Biol. Chem. 233, 184-188) and variously modified, was reinvestigated and applied to the measurement of submicrogram quantities of DNA in nuclear fractions and nucleic acid preparations. The reaction conditions were optimized using a small volume of DABA. This method measures 0.1 pg of DNA with a fluorescence twice that of background and is linear to 10 pg of DNA. DABA yields a lOOO-fold higher fluorescence with DNA compared with RNA, protein, and polysaccharides, and 0.1 pg of DNA is detectable in the presence of 200 pg of RNA or protein. The method is useful for detecting contaminating DNA in RNA preparations prior to hybridization. A simple procedure using ethanol precipitation was developed for removal of common interfering reagents such as sucrose, glycerol, salts, and Triton X-100. Nuclei isolated using detergents and assayed by this method are also free of measurable interfering lipids.
Methods
Reagents. Crude 3,5-diaminobenzoic acid was purchased from Gallard-Schlesinger (B2153) and purified as follows. The DABA (40 g) was dissolved in 200 ml of 4 N HCl. Norit A (1 g) was added, the mixture was