The Preparation of Fluorescein-Labeled Nucleic Acid Probes and Their Detection Using Alkaline Phosphatase-Catalyzed Dioxetane Chemiluminescence

A commonly used method for labeling nucleic acid probes nonradioactively is to introduce a small, relatively inert molecule, such as a hapten or biotin. Such molecules are generally coupled to a nucleoside triphosphate, such as dUTP, via a linker group. The modified nucleotide is then incorporated into the probe by an enzyme-mediated reaction, such as random prime labeling for long probes or terminal transferase-catalyzed 3′-end labeling for oligonucleotides. Following hybridization, detection takes place by means of an antibody (or, in the case of biotin, streptavidin) linked to an enzyme, such as alkaline phosphatase or horse-radish peroxidase, which is used to catalyze the detection reaction. A variety of haptens have been employed, most commonly digoxygenin, biotin, and fluorescein. This approach involves a longer detection protocol than a direct enzyme labeling methodology (see Chapter 10 ) because of the additional steps involved in membrane blocking, antibody binding, and subsequent membrane washes. However, it can be more readily adapted to high-sensitivity chemiluminescent detection with alkaline phosphatase-catalyzed dioxetane substrates. It can also be used with oligonucleotide probes, in this case coupled to horseradish peroxidase-catalyzed enhanced chemiluminescence detection, to take advantage of the rapid light output and low backgrounds of this reaction. This approach is described in Chapter 11 .