Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

Authors


  • The research activity of the Microbial Genetics group of the Department of Animal Biology and Genetics in the University of Florence, led by Prof. M. Polsinelli, is mainly focused on the biology of the nitrogen-fixing bacterium Azospirillum brasilense. Collateral to this research, one project is being developed that concerns the monitoring of soil bacteria in natural environment and their identification. Within this project, the work reported in the present paper was aimed at the production of probes to be used in the study of the ecology of Azospirillum in the rhizosphere. G. Damiani, from National Research Council in Milan, has been the first one in Italy to apply the RAPD technique.

M. Bazzicalupo.

Abstract

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.

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