Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Ribosomal RNA (rRNA) hybridization is crucial for phylogenetic classification.
• Existing methods like membrane-filter techniques face challenges due to rRNA secondary structure and variations in rRNA gene copy number.
• A more robust and accurate hybridization method is needed for large rRNA homology studies.

Purpose of the Study:

• To develop a new, improved method for ribosomal RNA hybridization.
• To overcome limitations of existing techniques, particularly those related to secondary rRNA structure and DNA complexity.
• To enhance the accuracy and reliability of phylogenetic classifications based on rRNA homology.

Main Methods:

• Synthesized complementary DNA (cDNA) on 25S rRNA fragments generated by mild alkali treatment.
• Enzymatically added polyadenylic acid tails to rRNA fragments.
• Hybridized rRNA tails with oligo deoxyribosylthymine and performed reverse transcription with tritiated TTP.
• Conducted hybridization reactions in solution and collected heteroduplexes on diethylaminoethylcellulose filter discs after S1 nuclease treatment.
• Utilized denaturation to avoid secondary rRNA structure issues before reverse transcription and hybridization.

Main Results:

• Achieved high percentages of duplex formation, ranging from 78-87%.
• Observed a low standard deviation in relative binding (averaging +/- 1.30%).
• Reported small differences in reciprocal hybridizations (1.71-5.18%).
• Successfully eliminated complications arising from variations in rRNA cistron numbers in nuclear DNA.

Conclusions:

• The novel rRNA hybridization method is highly efficient and reproducible.
• This technique effectively bypasses problems associated with secondary rRNA structure.
• The method offers superior accuracy and reliability compared to the membrane-filter technique for phylogenetic studies based on large rRNA homology.