Sequence analysis of trinucleotide repeat microsatellites from an enrichment library of the equine genome

Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsate llites have been elucidated. Thus, we constructed the equine genomic librar y enriched for DNA fragments containing (CAG)(n) repeats. The enriched meth od includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedure s are useful for the cloning of less abundant trinucleotide microsatellites . Microsatellites containing (CAG)(n) repeats were obtained at the ratio of one per 3-4 clones, indicating an enrichment value about 10(4)-fold, resul ting in less time consumption and less cost for cloning. In this study, 66 different microsatellites, (CAG)(n) repeats, were identified. The number of complete simple CAG repeats in our clones ranged 4-33, with an average rep eat length of 8.8 units. The microsatellites were useful as sequence-tagged site (STS) markers. In addition, some clones containing (CAG)(n) repeats s howed homology to human (CAG)(n)-containing genes, which have been previous ly mapped. These results indicate that the clones might be a useful tool fo r chromosome comparison between equines and humans.