Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic and chemiluminescent detection methods.
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.
Bierwerth
S
S
Department of Biology, University of Frankfurt, Germany.
Kahl
G
G
Weigand
F
F
Weising
K
K
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
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