Figure 2

cd -PCR based test. A. A schematic presentation of the test: primers flanking the deletion (D_A_3F/D_A_3R), combined with primers within the deleted sequences (CNGB3_4F/CNGB3_4R) were designed to amplify an affected and a normal chromosome, respectively. Primers flanking the deletion would not amplify a normal chromosome since they are more than 404 kb apart, but would give a product of 289 bp from an affected chromosome. The primer pair within the deletion would only amplify a normal chromosome and result in a 242 bp fragment. Dashed line represents the deleted region. B. cd-PCR based test results are observed in a colony pedigree genotyped for the deletion. A PCR product of 289 bp molecular weight is observed when using primers flanking the deletion (D-A-3 F/3R) in affected dogs (B1, dogs number 1 and 4), and in carrier dogs (B1, dogs number 2, 3, 5, 6, 7, 10, and 13). A PCR product is observed when using primers within the deleted sequence (CNGB3_4F/4R) in normal dogs (B2, dogs number 8, 9, 11, 12, 14, and 15) and in carrier dogs (B2, dogs number 2, 3, 5, 6, 7, 10, and 13). When using both primer pairs in a multiplex reaction, only one band of 289 bp long is observed in affected dogs (B3, dogs number 1 and 4), two bands are observed in carrier dogs (B3, dogs 2, 3, 5, 6, 7, 10, and 13) and only one band of 242 bp long is observed in the normal dogs (B3, dogs 8, 9, 11, 12, 14, and 15).