Description:
Phototope® Detection is based on chemiluminescence, an enzyme-catalyzed reaction which emits a blue light. This detection method is replacing radioactive detection due to sensitivity, convenience, safety and cost.
In the Phototope Detection Kits for nucleic acids, biotin associated with the target DNA provides the handle for the chemiluminescent detection. Biotinylated DNA is detected on a membrane support by first exposing the membrane to streptavidin, which binds to the biotinylated DNA. Next, biotinylated alkaline phosphatase complex is added, which binds to the streptavidin, resulting in the creation of a conjugate between the alkaline phosphatase and the DNA on the membrane. In the final step, the CDP-Star™ reagent is added. Alkaline phosphatase catalyzes the removal of the phosphate from the CDP-Star® (phenylphosphate substituted 1,2 dioxetane) to yield a moderately stable intermediate which then spontaneously decays, emitting light at 461 nm. The emitted light is detected by exposing the membrane to X-ray film for 1 to 10 minutes.
The biotin handle can be associated with your target DNA either directly (covalently) or by hybridization. Biotin can be incorporated directly into DNA by using enzymatic polymerization of DNA with a biotinylated primer (for DNA sequencing) or by polymerization in the presence of biotinylated nucleotide triphosphates (as with the NEBlot® Phototope® System).
Advantages:
Stability: Biotinylated probes and reagents are stable for extended periods unlike 32P-labeled probes which are only stable for a few days.
Speed: Only 40 minutes is required for the entire detection procedure. Exposure times of 1-10 minutes.
Multiple Exposures: Light is emitted at a constant rate for days, so you can perform multiple exposures to optimize signal intensity. Re-exposure at a future date is achieved by simply adding more chemiluminescent reagent.
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