โ Scribed by K.E. Selph; D.M. Karl; M.R. Landry
No coin nor oath required. For personal study only.
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 (\left.{ }^{3,7}\right]) 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 (\left(r^{2}\right)) 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 \mathrm{~h}). The method can reliably detect 17 amol of DNA ( (30 \mathrm{pg}) pUC 18 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. โฒ93 Academic Press, Inc.
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