Comparison of methods for the detection of in situ restriction enzyme – nick translation using fluorochromes and confocal microscopy

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Abstract
A series of experiments was carried out to determine the most efficient methods for detecting incorporated nucleotides in the "in situ" restriction enzyme – nick translation technique. Different methods were tested on fixed human metaphase chromosomes using confocal microscopy for the demonstration of the patterns produced. Of the various techniques tested, that using DIG-dUTP in conjunction with FITC-labelled anti-DIG appears to show the greatest sensitivity and specificity. The use of biotinylated nucleotides with FITC-avidin gives rather less sensitivity, while direct labelling with fluorescein-dUTP produces results more rapidly with better chromosome morphology but at the cost of reduced sensitivity. Resorufin-labelled dUTP was unusable, because of the low level of fluorescence and its very rapid fading. The successful fluorescence methods are more sensitive and faster than using horseradish peroxidase or alkaline phosphatase for detection.Key words: restriction enzymes, nick translation, chromosomes, fluorochromes, confocal microscopy.