A cloning strategy for identification of genes containing trinucleotide repeat expansions

Until today, nineteen trinucleotide repeat expansions larger than forty rep eat copies have been found in the human genome. Of these, the CAG/CTG repea t is predominant motif with twelve loci identified, ten of which have been associated with the development of neurodegenerative diseases. We have deve loped a cloning approach which isolates disease genes containing trinucleot ide repeat expansions. The method is based on size separation of genomic fr agments, followed by subcloning and library hybridization with an oligonucl eotide probe. Fractions and clones containing expanded repeats are identifi ed by the repeat expansion detection (RED) method throughout the cloning pr ocedure. Large family materials are not required and as little as 10 mug ge nomic DNA from a single individual is sufficient for this method. Using thi s strategy we have cloned two DNA fragments containing expanded repeats fro m two unrelated patients with a clinical diagnosis of cerebellar ataxia. Se quencing of the two fragments showed sequence identities with two disease g enes, the Huntington gene and the ataxin 3 gene, respectively. The method s hould be adaptable to the cloning of any long repeat motif in any species. Furthermore the experimental steps can be performed in less than a month, m aking it very effective and time efficient to disease gene identification.