Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms.
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.
Fani
R
R
Dipartimento di Biologia Animale e Genetica, Universitá degli Studi, Firenze, Italy.
Damiani
G
G
Di Serio
C
C
Gallori
E
E
Grifoni
A
A
Bazzicalupo
M
M
D000668:Ampicillin Resistance / Q000235:genetics; D016949:Azospirillum / Q000145:classification* / Q000235:genetics*; D015806:Azospirillum brasilense / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D016172:DNA Fingerprinting; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011110:Polymorphism, Genetic; D013302:Streptomyces / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D001616:beta-Galactosidase