Effects of Sucrose on Determination
of DNA by the
Method of Kissane and Robins
The fluorometric determination of DNA in nanogram amounts, as dcscribed by Kissane and Robins (l), has been widely applied (e.g., 2, 3, and 4). The method is based upon the reaction of DNA with solutions of 3,5-diaminobenzoic acid dihydrochloride (DABA) at elevated temperat'ures. Although the method, as described, is not sensitive to carbohydrates in the range normally found in tissues, we have found that sucrose, in the concentrations normally used for subcellular fractionations (e.g., 5), does interfere quite strongly with t.he determination of DNA. The present communication describes the extent as well as some of the properties of the interference, and also presents a method by which this interference can be avoided.
Materials and Methods. Highly polymerized calf-thymus DNA was obtained from Sigma (lot 86B-1680) and was prepared as described by Kissane and Robins (1). Highest purity sucrose was obtained from KEBO AB, Sweden. 3,&Diaminobenzoic acid was obtained from K & K Lbora'tories. The dihydrochloride (DABA) was prepa'red according to Kissanc and Robins (1).
The sample volumes used were 10 ,JL~, and contained DNA and/or sucrose as notIed below. The samples were treated with IO ~1 cold O.Bh: TCA. The ensuing extractions and washings (with alcoholic potassium acetate and absolute alcohol) were as described by Kissane and Robins (I), except that the volumes used for each step were 50 ~1, with approximately 45 ~1 being removed from the tube between steps. Variations from this procedure are noted below.
The reaction with DABA was carried out, by adding 10 ~1 of approximately 2 M DABA to the tubes containing the dried samples, and then heating the tubes at 60" for 30 mm in a shallow water bath. To each of the tubes, 150 ,pl 0.6 N PCA was then added, with mixing. An aliquot of 100 ,J was taken and diluted to 850 ,~l with 0.6 L%' PCA. The diluted fluorescent solution did not show any change in fluorescence readings for at least 2 hours.
Fluorescence was determined in an Aminco-Bowman spectrophotofluorometer. The sample was excited at 425 mp, and emission at 510 rnp