Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus.
Photobiotin was used to prepare biotinylated, nonradioactive nucleic acid probes for the detection of the RNA of barley yellow dwarf virus (BYDV) in plant extracts. A 1.7-kb cDNA of the PAV isolate of BYDV in the plasmid pUC8 vector was biotinylated and then used intact or as sonicated double-stranded DNA fragments. Simple methods were developed for the preparation of partially purified nucleic acid extracts of cereals and their spotting, after formaldehyde treatment, onto nitrocellulose membranes. After hybridization, biotin-labelled DNA bound to BYDV RNA on the nitrocellulose was detected with an avidin-alkaline phosphatase conjugate. BYDV RNA was readily detected with a sensitivity similar to that found with the same probe labelled with 32P by nick translation. Healthy plant extracts gave colourless spots.
Habili
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Department of Biochemistry, University of Adelaide, Australia.
McInnes
J L
JL
Symons
R H
RH
D000345:Affinity Labels; D001386:Azides; D001710:Biotin / Q000031:analogs & derivatives*; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics*; D005557:Formaldehyde; D001467:Hordeum / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*
D000345:Affinity Labels; D001386:Azides; D004274:DNA, Recombinant; D004279:DNA, Viral; D012367:RNA, Viral; D005557:Formaldehyde; D001710:Biotin; C045549:photobiotin