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F the g to a polymorphism in exon 6 is indicated for each individual. Dna extraction and sequence analysis ethylenediaminetetraacetic acid–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 5–18. 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 viagra no prescription order viagra online scams 2001). Human sequence (genbank accession numbers af305895–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 75–100 bp from the exons and all donor and acceptor splice sites were sequenced. Primer sequences and locations are listed in table 1. Exons 5–18 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.
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