Fractionation of nucleic acids isolated by the phenol- and DEP-methods from leaf tissues at different stages of development

Abstract

The phenol method and a new method based on the use of diethylpyrocarbonate (DEP) as a nuclease inhibitor were compared with respect to the yield of total nucleic acid extractable from barley and tobacco plants in different stages of development. The percentage distribution of different nucleic acid species in the extracts prepared by the two methods was also studied by sucrose density gradient centrifugation and MAK column chromatography. The nucleic acid yield in the extracts from first leaves of 11-day-old barley seedlings was higher with the DEP method than with the phenol method. With barley the same percentage distribution of the different nucleic acid species was obtained with both methods. By contrast, with tobacco (except for very young tissues) a preferential loss of hrRNA was observed when using the DEP method. The ageing of barley leaf tissues was characterized by a virtual increase in DNA apparently due to a preferential breakdown of rRNA. This pattern, characteristic of ageing tissues, was more pronounced with attached leaves than with detached, “artificially aged” tissues.