Having characterized the structure of DUP4 variants, we designed a simple robust junction fragment PCR assay that would allow detection of the DUP4 variant (both DUP4a and DUP4b) in nanograms of genomic DNA, at a large scale. This involved designing allele-specific and paralog-specific PCR primers across a known breakpoint, a process made more challenging by the high sequence identity between paralogs. DUP4-specific primers had a modified locked nucleic acid base incorporated in the terminal 3′ nucleotide to enhance specificity for the correct paralog.38 We initially targeted the GYPA-GYPB breakpoint that created the fusion gene but found that a similar breakpoint was present in a frequent gene conversion allele. We therefore designed primers to target the breakpoint between the GYPE repeat and the GYPB repeat, which was predicted to be unique to DUP4.