RAPID CLONING OF EXPANDED TRINUCLEOTIDE REPEAT SEQUENCES FROM GENOMICDNA

Trinucleotide repeat expansions have been shown to cause a number of n eurodegenerative diseases(1-6). A hallmark of most of these diseases i s the presence of anticipation, a decrease in the age at onset in cons ecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next(1). The involvement of trinucleotide repeat expansions in a number of other d iseases -including familial spastic paraplegia(7), schizophrenia(8), b ipolar affective disorder(9) and spinocerebellar ataxia type 7 (SCA7; ref. 10)-is suggested both by the presence of anticipation and by repe at expansion detection (RED)(10-12) analysis of genomic DNA samples. T he involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each exp ansion as a possible pathogenic mutation. We describe a novel procedur e for quick isolation of expanded trinucleotide repeats and the corres ponding flanking nucleotide sequence directly from small amounts of ge nomic DNA by a process of Repeat Analysis, Pooled isolation and Detect ion of individual clones containing expanded trinucleotide repeats (RA PID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected w ith ataxia and retinal degeneration.