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.
KeywordsMethylene Blue Probe Concentration Detection Procedure Whatman Paper Substrate Buffer
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