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|F the g to a polymorphism in exon 6 is indicated for each individual. Dna extraction and sequence analysis ethylenediaminetetraacetic acidâ€“stabilized blood from all individuals was available, and dna was extracted using a salting-out protocol (miller et al. 1988). The human chat gene has been mapped to hsa10q11. 2 (viegas-pequignot et al. 1991). A blastn search with the human mrna sequence against the canine genome showed the highest identity to cfa28 (96% identity, genbank accession number nw_876285. 1). The chat gene consists of 18 exons; r, n, m, s, and 5â€“18. Translation of the first 4 exons has not been described in animals, but a transcript including exon m and s have been amplified from a human spinal cord cdna library (ohno et al. can you buy viagra in uk kycoalcrafters.com viagra no prescription order viagra online scams kycoalcrafters.com 2001). Human sequence (genbank accession numbers af305895â€“af305906) was used to localize the exons in the canine genome, and primers for each exon were subsequently designed from intron sequence. The primers were located within a distance of approximately 75â€“100 bp from the exons and all donor and acceptor splice sites were sequenced. Primer sequences and locations are listed in table 1. Exons 5â€“18 representing the translated parts of the canine chat gene were polymerase chain reaction (pcr) amplified in 2 affected and 2 unaffected dogs. Each reaction consisted of 25 î¼l containing 2 î¼l of genomic dna, 10 pmol of each primer, 1. 5 mm mgcl2 (2. 5 mm mgcl2 for exon 15 and 16), and 0. 5 units of tempase hot start dna polymerase (ampliqon/bie & berntsen a-s, herlev, denmark). The cycling conditions were 1 cycle of denaturation at 95 â°c/10 min, followed by 35 three-segment cycles of amplification (95 â°c/30 s, 55 â°c/30 s, 72 â°c/30 s), and a final extension cycle at 72 â°c/10 min. The pcr products were purified by qiaquick pcr purification kit (qiagen, west sussex, uk) and, subsequently, sequenced on both strands. The same pcr primers and a bigdye terminator sequencing system were used according to.|