Generation Of Nonradioactive Probes

Environmental concerns, cost, and safety are a few reasons that nonradioactive alternatives to 32P-labeled probes are becoming more popular. Another advantage of nonradioactive probes is their long half-life, which permits them to be stored for extended periods of time. Several vendors provide kits for non-radioactive labeling and detection of probes, all of which employ the same basic concepts. Probes can be labeled by the traditional enzymatic methods (described earlier for radioactive probes), with the incorporation of alternatively labeled dNTPs or NTPs. Nonradioactive probes are typically labeled with haptens (such as digoxigenin, biotin, or fluorescein) (31-33). Detection of the hybridization signal depends on the type of label used, but it is either colorimetric or chemiluminescent.

Biotin-labeled probes are usually detected by enzyme-conjugated streptavidin and by enzyme-conjugated antihapten antibodies that detect hapten-labeled probes. The conjugated enzymatic activity (horseradish peroxidase or alkaline phos-phatase) results in the production of a colored precipitate in the presence of a specific chemical substrate (34), which allows direct visualization of hybridized bands on the membrane. However, colorimetric detection has several disadvantages that must be considered. Detection of rare sequences is limited and often requires long development times (>15 h). Furthermore, the colored precipitate cannot be efficiently removed from the membrane, thus preventing multiple uses of a single blot.

Chemiluminescent detection relies on the association of an enzyme-conjugated antibody with the digoxigenin, biotin, or fluorescein moieties of labeled probes. The reporter enzyme (horseradish peroxidase or alkaline phosphatase) dephosphory-lates a chemiluminescent substrate, thus generating an unstable anion that emits light as it decomposes (35). Positive signals are visualized by exposing the blot to X-ray film for short

Microsatellite Instability
Fig. 9. Generation of riboprobes.

lengths of time. Chemiluminescence does not produce a precipitate, which allows blots to be stripped and reprobed by standard methods. Chemiluminescent detection is sufficiently sensitive for most applications and is currently the most accepted alternative to radioactive probes. The major drawback to chemiluminescent detection is the frequent observation of high-background activity. However, strict adherence to the blocking steps provided in most protocols can significantly reduce background signal.

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