Endonucleolytic mutation analysis by internal labeling (EMAIL)

Mismatch-specific endonucleases are efficient tools for the targeted scanning of populations for subtle DNA variations. Conventional protocols involve 5'-labeled amplicon substrates and the detection of digestion products by LIF electrophoresis. A shortcoming of such protocols, however, is the limited 5'-signal strength. Normally the sensitivity of fluorescent DNA analyzers is superior to that of intercalating dye/agarose systems, however, pooling capacities of the former and latter approaches to mismatch scanning are somewhat similar. Detection is further limited by significant background. We investigated the activity of CEL nucleases using amplicon substrates labeled both internally and at each 5'-terminus. The amplicons were generated from exon 8 of the rice starch synthase IIa encoding gene. Signal of both 5'-labels was significantly reduced by enzyme activity, while that of the internal label was largely unaffected. In addition, background resulting from internal labeling was a significant improvement on that associated with 5'-labeling. Sizing of the multilabeled substrates suggests that 5'-modification enhances exonucleolytic activity, resulting in the removal of the dye-labeled terminal nucleotides. We have developed an alternative approach to mismatch detection, in which amplicon labeling is achieved via the incorporation of fluorescently labeled deoxynucleotides, which we have named Endonucleolytic Mutation Analysis by Internal Labeling (EMAIL). The strength of the EMAIL assay was demonstrated by the reclassification of a rice line as being heterozygous for the starch gene. This cultivar was assigned as being homozygous by a previous resequencing study. EMAIL shows potential for the clear identification of multiple mutations amongst allelic pools.