ABSTRACT
Many non-isotopic methods have been developed for nucleic acid detection, and recently some of these methods have been improved to the point where their sensitivity equals that of their isotopic counterparts. However, the robustness of these sensitive methods, usually based on alkaline phosphatase detection with 1,2-dioxetane substrates, tends to suffer from a high degree of signal-tonoise variability. Aside from this lack of robustness of the current methods, a further drawback arises from the inordinate amount of processing time involved in the procedure and the length of exposure time needed to obtain the desired sensitivity. These disadvantages undoubtedly contribute to the lack of wide acceptance of non-isotopic nucleic acid detection methods. We recently developed a complete system for the chemiluminescent detection of nucleic acids in Northern and Southern blot applications. This system combines a novel enhanced luminol substrate for horseradish peroxidase (HRP) with optimized hybridization and blocking steps that ensure consistent results with sensitivity equivalent to
P. This robust system also has the advantages of greatly reduced processing and film exposure time, and the high level of light output makes the system ideal for collecting data using cooled charge-coupled device (CCD) imaging systems. Posthybridization processing time has been reduced from the standard 2.5 to 1 h. Film exposure times range from 0.5 to 10 min with the substrate emitting light with relatively constant intensity over a 6-h period, thus allowing for multiple exposures.
