Nonradioactive Northern Blotting

Recently, nonradioactive techniques for detection of DNA and RNA have been developed (1 ,2 ). The most commonly used labels are “digoxigenin,” “fluorescein,” and “biotin” which are linked through a spacer to a nucleotide (in most cases UTP or dUTP) and are incorporated into specific gene-probes by various methods (e.g., random-priming, nick-translation, or PCR). The detection is based on the specific interaction of the labels with appropriate proteins, i.e., specific antidigoxigenin- and antifluorescein-antibodies or (strept)avidin, conjugated to alkaline phosphatase or peroxidase. The development of chemiluminescent substrates (e.g., CSPD, CPD, CPD-Star from Troptx) has improved the sensitivity of the detection to a range that was not achieved with calorimetric detection via chromogenic substrates (e.g., 5-chloro-4-bromo-indolylphosphate) for alkaline phosphatase.