An improved diagnostic test for rod cone dysplasia 1 (rcd1) using allele-specific polymerase chain reaction

To develop an improved diagnostic test for rod-cone dysplasia type 1 (rcd1). The rcd1 phenotype is an early onset, autosomal recessive disease caused by a mutation in the canine rod cyclic GMP phosphodiesterase beta-subunit (PDE6B) gene. A G to A transition in codon 807 at nucleotide position 2420 results in a stop codon. This is the only disease causing mutation detected so far in the canine PDE6B gene.

Allele specific primers were designed in which the 3 end had the nucleotide corresponding to either the wild type or the mutant rcd1 allele. PCR was done using the allele specific primers in combination with a common primer complementary to the opposite strand to distinguish between the wild type and the rcd1 alleles.

The wild type and rcd1 alleles were identified successfully in two independent ASPCRs done with two different sets of allele specific primers. Further, both alleles could be amplified in a single tube and distinguished based on the size difference of the PCR products using one allele specific primer of altered length by the addition of a 9 nucleotide long linker.

We have developed an improved diagnostic test for the disease based on ASPCR such that the presence or absence of different size amplified fragments provides direct determination of the genotype. In contrast to previously reported diagnostic tests, this method is more efficient because it eliminates the need for any further manipulation of the PCR product.