Abstract
Purpose: :
To characterize the length and gene content of this CNGB3 deletion, and develop a molecular test, to identify normal, carrier, and affected dogs both from individual samples and in pooled samples.
Methods: :
DNA was extracted from blood of normal and cd-affected dogs. Primers were designed, chosen initially within CNGB3 gene neighbors, to amplify genomic fragments to evaluate the size and identify the breakpoints of the deletion. PCR products were sequenced using ABI 3730 DNA analyzer, and analyzed using Sequencher 4.2.2. After establishing the breakpoints, a diagnostic test was designed and evaluated in pooled samples.
Results: :
11 primer pairs failed to amplify DNA of cd-affected dogs. Sequence analysis of PCR products revealed a 404,820 bp deletion. In addition to the entire CNGB3 gene, the deletion included parts of CPNE3 and CNBD1, resulting in a genomic fusion between these 2 genes predicted to cause loss of both gene products in affected dogs, as well as of CNGB3. Primers to identify normal, carrier and affected dogs were tested on known status dogs, and evaluated for pooling sensitivity. Serial dilution pools show that the primers detect 1 affected dog pooled with 19 normals.
Keywords: retina • genetics