IMPROVED TECHNIQUES FOR THE ENZYMATIC EXTRACTION OF NUCLEIC ACIDS FROM TISSUE SECTIONS

Abstract

Since the removal of only one of the two nucleic acids, ribonucleic and deoxyribonucleic, is often necessary in biological work, the optimum conditions for the specific removal of either nucleic acid by the corresponding enzyme were investigated. The specific removal of nucleic acids by enzyme treatment was tested by toluidine blue staining and by quantitative study of the radioautographic reactions produced by the labelled deoxyribonucleic acid and ribonucleic acid appearing in tissues of a cytidine-H3 injected mouse. For the removal of ribonucleic acid by ribonuclease, the tissues must be fixed in freshly prepared Carnoy's solution for 24 hours, as a shorter fixation permits the loss of ribonucleic acid from control tissue sections. Ribonuclease extraction (1 mg crystallized salt-free Worthington ribonuclease per 1 ml of distilled water) is carried out for 4 hours at 40°C. For the removal of deoxyribonucleic acid by deoxyribonuclease, it was also necessary to use 24 hour fixation in freshly prepared Carnoy. A high concentration of magnesium was found to be necessary in the incubation solution (0.05 mg crystallized Worthington deoxyribonuclease per 1 ml of 0.2 M Mg-containing Gomori's Tris maleic acid buffer solution). The incubation was carried out for 24 hours at 37°C. The toluidine blue stained basophilic material and the radioautographic reactions in the tissues of the cytidine-H3 injected mouse were lost following a double treatment with ribonuclease and deoxyribonuclease or by hot trichloracetic acid extraction. Therefore all the reactions appearing over the histological tissue sections are due to the tritium incorporated into deoxyribonucleic acid and ribonucleic acid. The basophilic material from cytoplasm and nucleolus was removed specifically and completely by ribonuclease treatment. The deoxyribonuclease treatment removed nuclear basophilia exclusively. However, controls of ribonuclease or deoxyribonuclease treatment showed no loss of basophilia, except on occasion in the pancreas. The radioautographic reactions found in the mouse tissues 24 hours after cytidine-H3 injection were exclusively localized over the nuclear basophilia of some nuclei after ribonuclease treatment, but were largely over the cytoplasmic basophilia after deoxyribonuclease treatment. Quantitative radioautographic study showed that either enzyme treatment removed its specific substrate completely and exclusively without loss in control preparations.