Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies.
In a new combination of techniques for ribosomal RNA hybridization, complementary DNA is synthesized on 25S ribosomal RNA fragments generated by mild alkali treatment, by the enzymatic addition of polyadenylic acid tails, hybridization of these tails with oligo deoxyribosylthymine, and reverse transcription in the presence of tritiated TTP. Hybridization reactions are performed in solution. Heteroduplexes are collected on diethylaminoethylcellulose filter discs after treatment with S1 nuclease. The problems presented by secondary rRNA structure are avoided by denaturation before reverse transcription and before hybridization. The high percentage of duplex formation (78-87%), the low standard deviation of relative binding (averaging +/- 1.30% relative binding), and small differences in reciprocal hybridizations (1.71-5.18% relative binding), as well as the elimination of complications resulting from differences in the number of rRNA cistrons in nuclear DNA, make this method preferable to the membrane-filter technique commonly used in phylogenetic classifications based on the homology of large rRNAs.
D004247:DNA / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D005796:Genes; D008722:Methods; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D012331:RNA, Fungal / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D015003:Yeasts / Q000235:genetics
D004271:DNA, Fungal; D009692:Nucleic Acid Heteroduplexes; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D004247:DNA