Evaluation of the biotinylated (Blugene) vs 32P-labeled cDNA probes of beta-glucocerebrosidase: relative sensitivities in genomic and other systems.
The sensitivity, rapidity, and ease of use of biotinylated (Blugene, Bethesda Research Laboratories) and 32P cDNA probes have been compared, the probe being the cDNA for beta-glucocerebrosidase (EC 3.1.2.45). With the Blugene kit I could detect 2 pg of biotinylated DNA on dot blots. However, under conditions of hybridization, the lower limit of detection for unlabeled cDNA (transblotted onto nitrocellulose) by its labeled counterpart was 5000-fold smaller (10 pg vs 50 ng) for the isotopically labeled probe. 32P- and Blugene-probes hybridized detectably with 0.5 and 10 micrograms, respectively, of transblotted EcoR 1-digested genomic DNA, making the radioactive method 20 times as sensitive. However, color development was complete within 30 min to 3 h, whereas radioautoradiography required 12 h to one week. Blugene was also safer, easy to use, and effective under appropriate conditions. The 32P method is expensive, hazardous, time-consuming, and technically difficult. This nonisotopic procedure represents a desirable improvement in biotechnology.
Department of Neurosciences, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
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Strasberg
D001710:Biotin / Q000032:analysis; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D015246:Deoxyribonuclease EcoRI; D004586:Electrophoresis / Q000379:methods; D005959:Glucosidases / Q000235:genetics*; D005962:Glucosylceramidase / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis; D010761:Phosphorus Radioisotopes / Q000032:analysis; D010957:Plasmids
D015342:DNA Probes; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D001710:Biotin; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D005959:Glucosidases; D005962:Glucosylceramidase