Home Microsatellite markers were developed as previously described or drawn from existing data sets [1-5]. Genotypes for linkage mapping were determined by PCR amplification of genomic DNA with a g32P end-labeled forward primer, as previously described [4,6].
A 3000 rad canine-hamster radiation hybrid panel RH08 was purchased from Research Genetics (Huntsville, AL). Markers were genotyped on the panel using standard protocols [3,7].
For both linkage and RH typing, PCR conditions were as follows unless otherwise noted: initial denaturation at 95°C for 1 min, followed by 35 cycles of 95°C for 20 s, Tm for 20s, and 74°C for 20s, followed by a final extension of 74°C for 5 min. Cycling conditions for Tm=Touchdown (TD) 65 were modified as follows: 10 cycles beginning at Tm=65°C and decreasing 1°C Tm per cycle, followed by 25 cycles at Tm=55°C.
Angio-associated migratory protein (AAMP) (GenBank accession no. M95627) was identified as a potential candidate for the cea gene based on its localization to human chromosome 2q35, the region corresponding to the cea candidate interval; putative involvement in angiogenesis and developmental regulatory pathways; and reported expression in human retinal tissue (UniGene cluster 83347).
Cloning utilized PCR primers derived from a mixture of the 1x dog genome sequence (exons 1, 3, and 6), and alignment of available canine and human gene sequence (Table C), generating initial partial sequence for exons 2, 4, 5, 7, 8, 9 and 11. Initial sequence used to clone and sequence the gene from a cDNA library (ZAP Express EcoRI/XhoI custom library, Stratagene, La Jolla, CA) derived from mature canine retina (Table D). The final canine reference sequence (Figure A) combines data from the canine 1x genome and cloned cDNA sequences, including complete coding regions and intron/exon boundaries of the AAMP gene.
To screen for mutations in AAMP, genomic DNA from one affected animal and one carrier were amplified by PCR, along with a corresponding negative control. Resulting amplicons were bidirectionally sequenced using Applied Biosystems ABI PRISM 3700 DNA Analyzer (Foster City, CA). Chromatogram files were aligned and analyzed using Phred (v. 0.000925, http://www.phrap.org), Phrap (v. 0990329, http://www.phrap.org), PolyPhred (v. 3.5, http://droog.mbt.washington.edu) and Consed (v. 11.0, http://www.phrap.org). Six single-nucleotide polymorphisms (SNPs) were identified among the introns and the 3'UTR of canine AAMP. SNP haplotypes were examined in two parents and 12 offspring from one of the experimental canine pedigrees and recombination events were observed between AAMP and cea, with multiple AAMP haplotypes segregating in phase with the cea disease allele. Thus, AAMP was excluded as a candidate for cea.
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