Labeling and colorimetric detection of nonisotopic probes

Although a number of different nonisotopic labels have been described in the literature, biotin and digoxigenin are used most frequently and are commercially available. Either label can be easily incorporated into DNA probes and be detected colorimetrically; a number of fluorochromes, as well as alkaline phosphatase and horseradish peroxidase (which produce colored precipitates) are available directly conjugated to anti-digoxigenin antibodies and to avidin. Chemiluminescent detection methods and indirect immunofluorescent techniques also provide sensitive alternatives for many molecular biology applications. This unit presents two protocols for incorporating biotinylated nucleotides into DNA probes by nick translation and random-primed synthesis. A Support Protocol describes colorimetric detection of the probes, which also serves to check the extent of nucleotide incorporation. An Alternate Protocol describes adaptations of the basic protocols for incorporation of digoxigenin-modified nucleotides.