Cross, MJ, Waters, DLE, Lee, SL & Henry, RJ 2008, ‘CEL I and the EMAIL Mutation Scanning Assay’, paper presented to International Plant and Animal Genome XVI Conference, San Diego, USA, 12-16 January.
Scanning for unknown mutations has been advanced by mismatch cleavage techniques. Pioneering methods include the use of ribonucleases, chemical cleavage of mismatch (CCM) and T4 endonuclease VII. Recently discovered enzymes, such as Endonuclease V from Thermotoga maritima and CEL I, have further advanced mismatch detection, due to a combination of useful properties. These include high specificity for a wide range of substrates, improved sensitivity for detecting a low level of mutants, and simple means of application. Standard approaches involve the PCR amplification of sample alleles using 5’-labeled primers and the size fractionation of digestion products by LIF electrophoresis. Such protocols, however, suffer from a limited ability to detect mismatch cleavage signal due to non-specific removal of the 5’-labeled termini. Mutation detection is further limited by high background that is characteristic of PCR-based mismatch scanning techniques. We have studied the activity of CEL nucleases using amplicon substrates labeled both internally and at each 5’ terminus. As nuclease activity increased, internal signal was maintained while 5' signal decayed. Furthermore, background from internal labeling was improved relative to 5' techniques. Observations of multi-labeled substrates suggest that CEL nucleases have a strong preference for 5’-labeled termini over mismatches. Non-specific removal of the 5’ labels appears to occur via exonucleolytic activity. An alternative mismatch scanning assay has been developed, in which amplicon labeling is achieved by PCR incorporation of fluorescently-labeled deoxynucleotides. We have named this strategy ‘Endonucleolytic Mutation Analysis by Internal Labeling’ (EMAIL). Multiple mutations amongst allelic pools have been detected when the EMAIL assay was combined with the nucleases CEL I and CEL II.