Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays.
A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.
Templeton
E F
EF
Kronem Systems Inc., Mississauga, Ontario, Canada.
Wong
H E
HE
Evangelista
R A
RA
Granger
T
T
Pollak
A
A
D000469:Alkaline Phosphatase / Q000032:analysis; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D012459:Salicylates / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D012459:Salicylates; D004247:DNA; D000469:Alkaline Phosphatase