title: Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic and chemiluminescent detection methods.
abstract: Digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI-restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P-labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear-cut fingerprints with high signal-to-background ratios within several minutes of exposure to X-ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin-based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to "equalize" band intensity differences. Whereas 32P-labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.
authors.affiliation: Department of Biology, University of Frankfurt, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Bierwerth
authors.forename: G
authors.initials: G
authors.lastname: Kahl
authors.forename: F
authors.initials: F
authors.lastname: Weigand
authors.forename: K
authors.initials: K
authors.lastname: Weising
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives; D003124:Colorimetry; D016172:DNA Fingerprinting / Q000379:methods*; D015342:DNA Probes; D005069:Evaluation Studies as Topic; D007887:Fabaceae / Q000235:genetics; D007211:Indoles; D008163:Luminescent Measurements; D003904:Mitosporic Fungi / Q000235:genetics; D009580:Nitroblue Tetrazolium; D009841:Oligonucleotides / Q000235:genetics; D010761:Phosphorus Radioisotopes; D010946:Plants, Medicinal; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D015342:DNA Probes; D007211:Indoles; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D000218:Adamantane