A sensitive method for detecting variation in copy numbers of duplicated genes

Gene duplications are common in the vertebrate genome, and duplicated loci often show a variation ill COPY number that may have important phenotypic effects. Here we describe a powerful method for quantification of duplicated copies based on pyrosequencing. A reliable quantification was obtained by amplification of the duplication breakpoint and a corresponding nonduplicated sequence in a competitive PCR assay. A comparison with all independent method for quantification based on the Invader technology revealed all excellent correlation between the two methods. The pyrosequencing-based method was evaluated by analyzing variation in copy number at the duplicated KIT/Dominant white locus in pigs. We were able to distinguish haplotypes at this locus by combining the duplication breakpoint test with a diagnostic test for a functionally important splice Mutation in the duplicated gene. An extensive allelic variation, including the presence of a new allele carrying a single KIT copy expected to encode a truncated KIT receptor, was revealed when analyzing white pigs from commercial lilies.