USE OF A DOT BLOT HYBRIDIZATION METHOD FOR IDENTIFICATION OF PURE TRANSFER-RNA SPECIES ON DIFFERENT MEMBRANES

The characterization of tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22-mu-m porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by cotrast, neutral 0.45-mu-m porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisae initiator tRNA(Met) in counter-current distribution fractions is shown.