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A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs
D K Lakshman1, S M Tavantzis, A Boucher
1Department of Plant Biology and Pathology, University of Maine, Orono 04469.
Analytical Biochemistry
|June 1, 1992
Summary
Researchers developed a new method to clone circular RNAs like viroids. This technique uses unique restriction sites to linearize and clone the circular cDNAs of plant pathogens for easier study.
Area of Science:
- Molecular Biology
- Virology
- Plant Pathology
Background:
- Viroids, virusoids, and satellite RNAs replicate via circular RNA intermediates.
- Cloning these circular RNAs presents unique challenges due to their structure.
Purpose of the Study:
- To develop a rapid and simple method for cloning naturally occurring viroids.
- To enable the cloning of sequence variants of well-characterized and novel plant RNA pathogens.
Main Methods:
- Surveyed unique restriction sites on circular cDNAs of viroids, virusoids, and satellite RNAs.
- Synthesized first-strand cDNA using random hexanucleotide primers and M-MuLV reverse transcriptase (Superscript RT).
- Synthesized second-strand cDNA via replacement synthesis using E. coli enzymes, linearized the circular double-stranded DNA, ligated it to a plasmid vector, and cloned in E. coli.
Main Results:
- Identified four unique restriction sites capable of linearizing circular cDNAs.
- Successfully developed and demonstrated a cDNA cloning procedure for a viroid from Nematanthus wettsteinii.
- The method is suitable for various circular RNA plant pathogens.
Conclusions:
- A novel and efficient cDNA cloning method for circular RNA plant pathogens has been established.
- This technique facilitates the study of sequence variants and new pathogens.
- The method offers a valuable tool for viroid and satellite RNA research.