Quantification of chemiluminescent DNA probes using liquid scintillation counting.
A novel method for quantifying chemiluminescent DNA probes is described. The method uses liquid scintillation counting to measure light emission from the alkaline phosphatase-catalyzed breakdown of the substrate PPD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1 (3.7[decan]- 4-yl)phenyl phosphate) on dot blot preparations. Serial dilutions of either pUC18 DNA or lambda DNA were hybridized with digoxigenin-labeled probes and detected using the method described. Light flux (luminescence) was linearly related to DNA concentration, typically with a coefficient of determination (r2) of 0.9 or better. Due to the stability of alkaline phosphatase and the long-lived luminescence of PPD in the Lumi-phos formulation, repetitive analyses of a given sample can be made for up to 20 h. The method can reliably detect 17 amol of DNA (30 pg pUC18DNA) with a coefficient of variation on replicate samples of 14%. Optimization experiments showed that 7% sodium dodecyl sulfate in the prehybridization and hybridization buffers resulted in the lowest background; the best combination of signal-to-noise ratio and reproducibility was obtained using Bio-Rad Zeta-Probe GT nylon membranes. Direct immersion of samples into a solution of substrate was found to give the most precise results and ensured that substrate limitation at high concentrations of alkaline phosphatase (i.e., higher DNA amounts) did not occur.
School of Ocean and Earth Science and Technology, Department of Oceanography, University of Hawaii, Manoa, Honolulu 96822.
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Karl
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Landry
D000218:Adamantane / Q000031:analogs & derivatives / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D003720:Densitometry; D004076:Digoxigenin; D005456:Fluorescent Dyes / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008027:Light; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009757:Nylons; D012588:Scintillation Counting / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D009757:Nylons; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D000469:Alkaline Phosphatase; D004076:Digoxigenin; D000218:Adamantane