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[3.3.1.13,7]decan]-4- yl)phenyl phosphate) on dot blot preparations. Serial dilutions of either pUC18 DNA or λ 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 pUC18 DNA) 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. © 1994 Academic Press, Inc. All rights reserved.