A Method of Quantitative Determination of Nucleic Acids in Plant Cells

Abstract

After several model experiments, we modified Schmidt and Thannhauser's method (Schmidt and Thannhauser, 1945) for the quantitative determination of nucleic acids and applied this modified method to plant cells. Procedure of the method is as follows:
1. Separation: A piece of plant tissues is weighed and ground for about 10 minutes in ca. 10 volumes of ice-cold 7% trichloroacetic acid. The suspension is centrifuged. The above treatment is repeated 3 times. Supernant contains acid soluble phosphorus substances. The residue is washed succesively with an ice-cold soln of 1% trichloroacetic acid, ice-cold water and ethanol. Then the residue is suspended in 30-40 volumes of mixture of ethanol and ether (3:1) and boiled for 5 minutes. Centrifuged. The residue is washed with ether followed by boiling for 30 minutes in 30-40 volumes of methanol-chloroform mixture (1:1). After centrifugation the residue is washed with ether. Phospholipids are extracted by the above treatment with organic solvents. The residue is dried and suspended in N-KOH (10ml per gram of fresh tissues) for 15 hours at 37°C. Centrifuged. The supernant is removed, 0.2 volumes of 6N-HCl and one volume of 5% trichloroacetic acid are added to one, volume of supernant, and kept in an ice-box for 30 minutes. White ppt is obtained. Then the suspension is centrifuged. Most of DNA is contained in ppt while phosphoproteids (in decomposed form), PNA (pentosenucleic acid) and a small amount of DNA are found in the supernant. The amount of DNA contained in the supernant is measured with Dische's method. PNA and inorganic P derived from phosphoproteids are separated by Delory's method (Delory, 1938) modified by us.
2. Decomposition: The result of our model experment showed that the decomposition of organic substances with perchloric acid gave better result than to ignite them over a microburner with H₂SO₄ or HNO₃ (cf. King, 1932). Fig. 2. Absorption of developed color at different concentrations of phosphorus below 1.0γ/ml Fig. 3. Absorption of developed color at different concentrations of phosphorus between 60γ/ml and 100γ/ml
3. Reagent: a) 50% HNO₃, b) 10% ammonium molybdate soln, c) ammonium vanadate soln (Dissolve 1.175g of ammonium metavaradate in 10ml conc. HNO₃ and add 460ml dist. water).
4. Determination: For the quantitative determination of P, a method recommended by Fiske and Subbarow (1925) has frequently been employed by several investigators. But the result of our experiments showed that the color developed by this method was not stable and that a modified Barton's method (Barton, 1948) was preferable to the method of Fiske and Subbarow. In this phosphovanadomolybdate method (PVM method), the color developed is very stable and is hardly affected by the presence of silicates (Shigematsu, unpubl). Procedure: 0.6ml of reagent a) 1.0ml of reagent b) are added to 5ml of sample soln and followed by the addition of 3.4ml of reagent c). Keep 10 minutes at room temperature. Orange color developes. The transmittance of the color at different wavelengthes is shown in Fig. 1. In this figure it is seen that a maximum spread between the blank and the color developed is at 405mμ-410mμ. This wavelength is used for the spectrophotometric determination with a Beckman's spectrophotometer. The absorbance is proportional to the concentrations of P between 0.3γ/ml and 80γ/ml (Figs. 2 and 3).