Deoxyribonucleic acid-ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids.
A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.
D002021:Buffers; D002482:Cellulose; D004269:DNA, Bacterial; D004121:Dimethyl Sulfoxide; D004926:Escherichia coli / Q000032:analysis; D005374:Filtration; D005559:Formamides; D005827:Genetics, Microbial; D006824:Hybridization, Genetic; D008722:Methods; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D012270:Ribosomes / Q000032:analysis*
D002021:Buffers; D004269:DNA, Bacterial; D005559:Formamides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D002482:Cellulose; D004121:Dimethyl Sulfoxide