Cloning of canine CNGB3 cDNA. The canine CNGB3 gene was obtained by screening a canine retinal cDNA library (Stratagene, La Jolla, CA) using a PCR based method described previously [Wang, 1996 #2259]. PCR primers CNGB3-2 and CNGB3-7 (Table 2) were designed based on human CNGB3 sequence (GenBank accession no. AF272900). PCR amplification reactions were performed using 0.4 µM of each primer in a 25 µl reaction containing total cDNA prepared as described [Wang, 1996 #2259], 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.2 mM each of dATP, dCTP, dGTP and dTTP, and 2.5 units AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). After an initial denaturation at 94°C for 3 min, samples were amplified for 30 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a final extension of 72°C for 7 min. PCR products were separated by gel electrophoresis, isolated, purified by Qiagen column, and then directly sequenced using an ABI 377 automated sequencer (Applied Biosystems, Foster City, CA).

The 5’ end of the gene was cloned using canine specific 5’ reverse primer CNGB3-16 and vector primer PBK-III(f) (Table 2) in an amplification reaction as described above. Similarly, canine specific 3’ forward primer CNGB3-19 was used in combination with the vector primer PBK-VI(r) to obtain the 3’ end of the gene.

The full length canine CNGB3 cDNA (GenBank accession no. AF490511) was amplified as three overlapping regions using primers CNGB3-EX1F and CNGB3-16, CNGB3-20 and CNGB3-21 and CNGB3-19 and CNGB3-EX18R (Table 2A). A partial CNGB3 cDNA was amplified for use as a probe for Northern analyses and BAC library screening using primers CNGB3RT-EX5 and CNGB3RT-EX7R (Table 2A).

The amino acid translation of canine CNGB3 was aligned with corresponding human and mouse sequences by MegAlign, part of the DNASTAR software package (DNASTAR, Inc. Madison, WI), using CLUSTAL W (version 1.8) [Thompson, 1994 #2289].

BAC clone isolation. An arrayed canine 8.1 fold BAC library [Li, 1999 #705] was probed with a partial CNGB3 cDNA probe generated by RT-PCR using primers CNGB3RT-EX5F and CNGB3RT-EX7R as described above. A CNGB3-positive BAC clone (100-O2) was isolated, purified by alkaline lysis, NotI digested and sized separated on a FIGE mapper (BioRad, Hercules, CA) to determine the size of the BAC 100-O2 insert (140 kb). BAC ends were sequenced utilizing vector (pBACe3.6, GenBank accession no. CVU80929) specific Sp6 and T7 promoter sequence primers. From the two end sequences of BAC 100-O2, two STS markers were developed utilizing primer pairs: 100-02-TJ-1, 100-02-TJ-2; and 100-02-TV-1, 100-02-TV-2 respectively (Table 2B). To test for presence of the BAC-end STSs PCR reactions utilized genomic DNA from cd/+ cd/cd and +/+ animals.

"Shotgun" sequencing of the BAC 100-O2 insert to 3x was undertaken by The Institute for Genomic Research (Rockville, MD). Briefly, the fragmented BAC DNA was cloned into the pBR322 based vector pHOS2 (unpublished vector) containing BstXI adapters and electroporated into DH10b E.coli (Invitrogen Life Technologies, Carlsbad, CA). Clones containing 1.8-2 kb DNA fragments were randomly picked and grown overnight in YT media, and plasmid DNA extracted and sequenced. 784 sequences, with an average of 700 nucleotide length were generated and assembled using the phred/phrap software (http://www.phrap.org/) resulting in 19 unordered contigs. The contigs were masked for repetitive elements using RepeatMasker (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker/) and compared against non-redundant (nr), EST (dbEST) and high-throughput genomic sequence (htgs) GenBank nucleotide databases using BLAST@CBSU (http://ser-loopp.tc.cornell.edu/cbsu/index.htm) software.

Exons 1-3 and their intron/exon boundaries were not present in the shotgun library generated from BAC 100-O2. Intron 1 was amplified by Long Range PCR using ELONGASE Enzyme Mix (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s guidelines utilizing primers CNGB3-EX1F and CNGB3-EX2R (Table 2C). We attempted unsuccessfully to clone introns 2 and 3. The corresponding region in the human for intron 2 is ~13,000bp and for intron 3 is ~55,000 bp. The sizes for introns 2 and 3 are believed to be similar in dog. Using BAC 100-02 DNA as a template, exons 2 and 3 were PCR-amplified using primers CNGB3–EX2F and CNGB3-EX2R and CNGB3-EX3F and CNGB3-EX3R (Table 2C) respectively. It should be noted that primers CNGB3-EX2R, CNGB3-EX3F and CNGB3-EX3R anneal to the coding sequence of exons 2 and 3.

Primers CPNE3-f1 and CPNE3-r1 (Table 2) were also designed to amplify a 215 bp STS corresponding to a fragment of BAC 100-O2 that represents a highly conserved non-coding region in the 3' UTR (exon 12) of the canine homolog of CPNE3 (Copine 3, MIM 604207). PCR conditions were as described above.

CNGB3 Exon scanning in GSPs. Genomic DNA isolated from affected GSPs was scanned for CNGB3 mutations. For exons 1 and 4-18, primers were designed to amplify each exon plus about 50 bp of flanking intronic sequence. For exon 2, CNGB3-EX2R anneals to the last 20 bp of the exon. Similarly, CNGB3-EX3F anneals to the first 20 bp and CNGB3-EX3R to the last 20 bp of exon 3. PCR reactions were carried out in 25 µl volumes containing 200 ng genomic DNA, 0.4 µM of each primer pair in a volume of 25 µl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3) and 0.2 mM each of dATP, dCTP, dGTP and dTTP and 2.5 units AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). After initial denaturation at 94°C for 3 min, the samples were amplified for 30 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec with the final extension of 72°C for 7 min. PCR products were electrophoresed and sequenced.

Exon 6 Allele Discrimination Test. PCR amplification utilizing primers CNGB3-EX6F and CNGB3-EX6T (Table 2) were carried out in 25 µL volumes containing 200 ng genomic DNA, 0.4 µM of each primer pair in a volume of 25 µl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3) and 0.2 mM each of dATP, dCTP, dGTP and dTTP and 2.5 units AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). After initial denaturation at 94°C for 3 min, the samples were amplified for 30 cycles at 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec with the final extension of 72°C for 7 min.

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