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.
Selph
K E
KE
School of Ocean and Earth Science and Technology, Department of Oceanography, University of Hawaii, Manoa, Honolulu 96822.
Karl
D M
DM
Landry
M R
MR
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