Figure 1. Strategy for cloning the canine PDEalpha cDNA

The cDNA containing the coding sequence (shaded box), the untranslated regions at the 5'- and 3'-end (open boxes) and two sites where poly (A) are added, is shown as an insert in the vector pBK-CMV (Stratagene, La Jolla, CA) used for the construction of the library. Clones containing different overlapping regions of the cDNA (1-PDEA, 2-PDEA, 5'-PDEA, 3'-PDEA, PDEA-S and PDEA-L), the sizes of the cDNA fragments in the clones, and the primers (see Table 1 for sequence) used for generating those fragments are identified. The clones 1-PDEA and 2-PDEA were obtained by reverse transcription and polymerase chain reaction (RT-PCR) from canine retinal total RNA using the RT-PCR kit (Perkin-Elmer; Foster City, CA) as recommended by the manufacturer. Amplification of cDNA to obtain 1-PDEA and 2-PDEA was done for 30 cycles at 94 °C (1 min), 54 °C (2 min), 72 °C (2 min). Other clones (5'-PDEA, 3'-PDEA, PDEA-S and PDEA-L) were obtained by screening a canine retinal cDNA library using a PCR based method. The PCR for screening the canine retinal cDNA library was done in 50 μl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.2 mM dNTPs, 2.0 mM MgCl₂, 10% DMSO, and 1.25 units Taq polymerase (Life Technologies; Grand Island, NY). The conditions for PCR amplification of each fragment were as follows: 5'-PDEA and 3'-PDEA were obtained by 30 cycles at 94 °C (1 min), 60 °C (1.5 min), 72 °C (2 min); PDEA-S and PDEA-L were obtained by 30 cycles at 94 °C (1 min), 58 °C (2 min), 72 °C (3 min). All reactions were concluded with a single step extension reaction at 72 °C for 10 min.