Using non-radioactive probes on plants: a few examples.
Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a post in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes.
Accotto
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Istituto di Fitovirologia Applicata (CNR), Torino, Italy.
Vaira
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AM
Noris
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Vecchiati
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D000218:Adamantane / Q000031:analogs & derivatives; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D004279:DNA, Viral / Q000032:analysis*; D017791:Geminiviridae / Q000235:genetics / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D018551:Solanum lycopersicum / Q000821:virology*; D010935:Plant Diseases; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis
D004279:DNA, Viral; D007202:Indicators and Reagents; D012333:RNA, Messenger; D011994:Recombinant Proteins; C071872:3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D000218:Adamantane